
Book 1^ 5^ 
Copyright NU334- 



CJ2EYRIGHT DEPOSIT. 



ELEMENTARY EXERCISES 



IN 



PHYSIOLOGY 



BY 



PIERRE A. FISH, D.Sc, D.V.M. 



PROFESSOR OF VETERINARY PHYSIOLOGY 

NEW YORK STATE VETERINARY COLLEGE 

CORNELL UNIVERSITY 



Fourth Edition Revised 



COMSTOCK PUBLISHING CO., AGENTS 

ITHACA, N. Y. 

1921 






COPYRIGHT, 1921 

BY 
PIERRE A. FISH 



W4r 18 1221 

©CLA617065 
<2sU , h 



Preface to the Fourth Edition 



The purpose of instruction is to fix ideas and facts in the brain 
where they may be available when needed. Information conveyed 
to the brain through the eye is a very direct route, for a thing once 
seen is more easily remembered than any amount of verbal descrip- 
tion of it. This is the important function of a laboratory course. 
An experiment carried through its various details understandingly 
can be visualized by the observer with less tax upon his mental 
powers than by any other method. 

It has been the aim in this course to select experiments which 
are elementary but fundamental in the physiological processes of 
the animal body, in order that they may serve as a basis for ad- 
vanced work and for a clearer conception of the importance of a 
knowledge of the normal mechanism in its relation to pathology and 
medicine rather than a consideration of physiology as an isolated 
subject. 

The laboratory more than any other method trains the power of 
observation; cultivates skill and accuracy and emphasizes the fact 
that first hand knowledge is the most desirable. 

In the preparation of this guide, numerous standard books and 
papers have been drawn upon, in addition to which a number of 
useful suggestions from assistants, past and present, have been in- 
corporated. 

P. A. F. 

January 1921. 



PART I 



Chemical Physiology 



TABLE OF CONTENTS 



PART I 



List of Apparatus for Chemical Physiology. List of Reagents. 
General Directions. Foods. 

Chapter I 

Proteins; Classification; Albumins; Xanthoproteic, Millon's, 
Piowtrowski 's Reaction ; Ferrocyanide and Sodium Sulphate Tests ; 
Indiffusibility of Albumin; Globulins; Serum Globulin; Myosin- 
ogen ; Vitellin ; Peptone pp. 18-24 

Chapter II 

Derived Proteins ; Acid and Alkali Albumins or Albuminates ; 
Syntonin ; Albuminates ; Metallic Albuminates ; Coagulated Pro- 
teins ; Proteoses or Albumoses ; Hemoglobins ; Glycoproteins ; Nu- 
cleoproteins ; Keratin, Elastin, Collagen, Gelatin Tests; Bone. 

pp. 25-29 
Chapter III 

Carbohydrates; Monosaccharids ; Disaccharids; Polysaccharids ; 
Starch ; Glycogen ; Dextrin ; Cellulose pp. 29-32 

Chapter IV 

Dextrose; Trommer's Test; Fehling's Solution; Boettger's Test; 
Phenyl-hydrazine Test; Yeast Test; Barfoed' Reagent; Lactose; 
Koumiss; Kephyr ; Sucrose ; Maltose pp. 32-36 

Chapter V 

Fats ; Lipase ; Glycerin ; Fatty Acids ; Saponification ; Neutral 
Fats; Stearin; Palmitin; Olein; Emulsion pp. 36-38 

Chapter VI 
Examination for Proteins and Carbohydrates pp. 38-39 

Chapter VII 
Salivary Digestion ; Reaction ; Ptyalin ; Mucin ; Albumin ; ; 
Sulphocyanide of Potassium; Nitrites; Chlorides; Sulphates; 



Starch ; Soluble Starch ; Erythrodextrin ; Achroodextrin ; Maltose ; 
Effect of Drugs on Salivary Digestion; Bread; Potato. . .pp. 39-42 

Chapter VIII 

Gastric Digestion ; Reaction ; Pepsin ; Hydrochloric Acid ; 
Rennin ; Fibrin ; Gelatin pp. 43-44 

Chapter IX 

Syntonin (acid albumin) ; Proteose (albunfose) ; Peptone; Biuret 
Reaction; Effect of Drugs on Gastric Digestion; Rennin; Gastric 
Lipase pp. 44-47 

Chapter X 

Pancreatic Digestion ; Trypsin ; Amylase ; Lipase ; Milk Curd- 
ling Enzyme ; Secretin ; Pancreatin; Alkali-Albumin; Albumose ; 
Peptone ; Indol pp. 47-49 

Chapter XI 

Emulsion Experiment ; OleicAcid ; Olive Oil ; Action on Fat 
Neutral Oil ; Action on Milk ; Amino-Acids ; Co-enzyme ; Erepsin 
Succus Entericus ; Invertase ; Maltase ; Lactase ; Cane Sugar 
Maltose pp. 49-53 

Chapter XII 

Bile, Reaction ; Glycoholic Acid ; Taurocholic Acid ; Soaps ; 
Lecithin; Cholesterin; Bilirubin; Biliverdin; Stereobilin; Mucin; 
Iron; Pettenkof er 's Test ;Gmelin's Test; Hay's Test; Bile in Di- 
gestion ; Emulsion pp. 53-56 

Chapter XIII 

Milk; Reaction; Analysis of Human and Cow's Milk; Cream; 
Specific Gravity; Fat Globules; Lactalbumin; Milk Sugar (Lac- 
tose) ; Curdi; Whey; Rennin; Casein; Para-casein; Calcium Salts; 
Galactomicrons pp. 56-62 

Chapter XIV 

Blood; Specific Gravity; Red and White Corpuscles; Hemo- 
globin ; Laky Blood ; Defibrinated Blood ; Indiffusibility of Hemo- 



globin; Hemin; Coagulation; Serum; Plasma; Fibrin; Buffy Coat: 
Salted Plasma; Oxalate Plasma; Fibrin Ferment pp. 63-66 



Chapter XV 

Protein Reactions of Blood ; Chlorides ; Sulphates ; Phosphates ; 
Dextrose; Odor of Blood; Paraglobulin ; Fat; Blood Ash; Iron; 
Spectroscope ; Oxyhemoglobin ; Reduced Hemoglobin; Stokes' 
Fluid ; Chylomicrons pp. 66-69 

PART II 

Chapter XVI 

Dissection of Frog's Heart; Pericardium; Ventricle; Auricle; 
Truncus Arteriosus ; Frenum ; Carotid Artery ; Pulmo-Cutaneous 
Artery; Aorta ; Venae Cavae; Sinus Venosus; Semi-lunar Valves: 
Spiral Valve ; Pylangium ; Synangium ; Vagus Nerve ; Glosso- 
pharyngeal Nerve ;Sartorius Muscle ; Adductor Magnus ; Adductor 
Longus; Rectus Internus Major (Gracilis) ; Rectus Interims Minor; 
Triceps Extensor Femoris ; Rectus Anticus Femoris ; Vastus In- 
ternus; Vastus Externus; Gluteus; Biceps; Semi-membranosus ; 
Pyriformis ; Semi-tendinosus ; Adductor Brevis ; Peetineus ; Ilio- 
*psoas; Quadratus Femoris; Obturator; Gastrocnemius; Tibialis 
Posticus ; Tibalis Anticus ; Extensor Cruris ; Peroneus ; Sciatic 
Nerve pp. . 71-77 

Chapter XVII 

Lymph Hearts ; Pithing Frog : Number of Heart Beats ; Heart 
Tracing ; Ciliary Motion pp. 77-78 

Chapter XVIII 

Circulation of Blood; Arteries; Capillaries; Veins; Axial and 
Peripheral Zones; Red and White Corpuscles; Inflammatory Con- 
ditions; Migration of Leucocytes; Diapedesis pp. 79-82 

Chapter XIX 

Reflex Action; Effect of Mechanical Stimuli; Effect of Acids; 
Shock; Strychnine; Chloral Hydrate; Nerve Muscle Preparation; 
Galvani's Experiment ; Demarcation Currents pp. 82-83 



Chapter XX 

Inductorinm ; Primary and Secondary Coils; Electrical Field; 
Short Circuiting Key; Electrodes; Battery; Carbon; Zinc; Make 
and Break Shocks; Maximal and Minimal Shocks; Interrupted 
Shocks; Kathode; Anode; Electrolysis of Potassium Iodide; Break 
Extra-Current j Rheocord; Unipolar Excitation; Polarization of 
Electrodes pp. 84-91 

Chapter XXI 

Mechanical, Thermal, Chemical and Electrical -Stimuli ; Make 
and Break Currents ; Maximal and Minimal Stimuli ; 'Single Induc- 
tion Shocks ; Constant Current ; Interrupted Current ; Nerve Im- 
pulse and Electrical Impulse Not Identical pp. 91-93 

Chapter XXII 

Secondary Contraction; Secondary Tetanus; Secondary Con- 
traction from Nerve ; Secondary Contraction from the Heart ; 
Paradoxical Contraction; Electrotonus ; Experiment with Ergo- 
graph pp. 93-96 

Chapter XXIII 

Elasticity and Extensibility of Muscle ; Independent Irritability 
of Muscle; Bernard's Method; Relative Excitability of Muscle and 
Nerve ; Changes in the Excitability of a Nerve When Dying ; Dead 
Muscle and Nerve; Kuehne's Sartorius Experiment pp. 96-99 

Chapter XXIV 

The Moist Chamber; The Muscle Curve; Amplification 'or Mag- 
nification ; Work Done During a Single Contraction ; Record of the 
Thickening of a Muscle pp. 99-103 

Chapter XXV 

Influence of Veratrine on the Contraction of Muscle ; Fatigue of 
Muscle; Tetanus pp. 103-105 

Chapter XXVI 

Influence of Temperature upon the Contraction of Muscle ; In- 
fluence of Load upon the Contraction of Muscle ; Isotonic ; Isometric ; 



After Load ; Induction in Nerves ; Telephone Experiment ; Form 
and Volume of Contracting Muscle pp. 105-108 

Chapter XXVII 

Cardiac Vagus ; Stimulation ; Latent Period ; Inhibition ; Escape ; 
Action of Drugs on Heart ; Muscarine ; Atropine ; Pilocarpine ; 
Nicotine pp. 108-110 

Chapter XXVIII 

Sphygmographs ; Ludwig's ; Von Frey 's ; Richardson 's ; Teske's ; 
Sphygmomanometers; Pneumograph pp. 110-116 

Chapter XXIX 

Artificial Scheme of the Blood Circulation ; Conversion of an 
Intermittent Flow into a Continuous Flow ; Relation of Peripheral 
Resistance to Blood Pressure ; Pulse ; Mean Pressure ; Gravity ; 
Lymph Circulation pp. 116-121 

Chapter XXX 

Blood Pressure in the Frog; Stannius' Experiments on the 
Frog's Heart; Cardiac Delay or Latent Period of Cardiac Muscle; 
Maximum Contractions Only ; Staircase Contractions of the Heart ; 
Location of Motor Centers in Frog's Heart; Effect of Temperature 
Upon Heart Beat; Isolated Apex; Intracardiac Inhibitory Center 
in Frog pp. 121-126 



CHEMICAL PHYSIOLOGY 



APPARATUS FOR THE LOCKER 



1 dozen test tubes, 6 inch 

1 dozen test tubes, 5 inch 

1 minim pipette 

1 Beaker, 10 oz. 

1 Graduate, 30 mils 

1 Graduate, 250 mils. 

1 Flask 

1 Funnel, 1% inch 

1 Funnel, 3 inch 

1 Watch glass 

1 Evaporating dish, 8 oz. 

1 Urinometer 

1 Glass rod 

1 Dialyzer 

1 Thermometer 

1 Crucible, 8 mils 



1 Piece wire gauze 

1 Piece absorbent cotton 

1 Box matches 

1 Test tube brush 

1 Test tube rack 

1 Test tube holder, wire 

1 Tripod 

1 Piece muslin 

1 Pack filter papers, 3 inch 

1 Pack filter papers, 6 inch 

1 Sponge 

1 Clay triangle 

2 Tin cans 

1 Copper water bath 
1 Towel 



Special apparatus, not found in the locker, may be obtained, when needed, 
by handing an order for it to one of the assistants. 



EXPERIMENTAL PHYSIOLOGY 



APPARATUS 


1 Metal tray 




1 Frog board with 4 clips 




1 Wooden stand 


Battery 


1 Iron standard with 2 clamps 


Induction coil 


1 Femur clamp 


Make and break key 


1 Muscle lever 


Short circuiting key 


1 Strip of pins 


Electrodes 


5 Coils of wire 


Kymograph 


5 Sheets Kymograph paper 




Saline solution 





Special apparatus, not found in the locker, may be obtained, when needed, 
by handing an order for it to one of the assistants. 



LIST OF REAGENTS 



Ammonium oxalate solution: saturated. 

Barfoed's reagent: 

Cuprie acetate 13 grains 

Distilled water 200 mils * 

Acetic acid (38% ) 5 mils 

Barium chloride solution: 2% 

Baryta mixture: 

Saturated solution of Barium nitrate 1 volnme 

'Saturated solution of Barium hydrate 2 volumes 

Basic lead acetate or liquor plumbi subacetatis 

Lead acetate 17 grams 

Lead oxide 10 grams 

Distilled water 80 mils 

Boil half an hour. Let co'ol and add enough previously boiled distilled 
water to make 100 mils. 

Biuret reagent, (Gies): 

10% -Sodium hydroxide 1000 mils 

3% Cuprie sulphate 25 mils 

Calcium chloride solution: 2 % 

Copper sulphate solution: 1 % 

Fehling's solution: 

A. Pure copper sulphate 34.64 grams 

Distilled water, to 500. mils 

B. Sodio-potassium tartrate (Bochelle salts) 173. grams 

Potassium hydroxide 125. grams 

Distilled water, to •. 500. mils 

Keep in separate bottles. When ready to use, mix equal parts of A. and B. 

Hydrochloric acid: 0.2%. May be made by adding 6.5 mils of the concen- 
trated acid to 1 liter of water. 

Iodine solution: (may be diluted if necessary) 

Iodine 1 gram 

Iodide of potassium 2 grams 

Distilled water 300 mils 



* In accordance with the recommendation of the last edition of the Pharma- 
copoeia the term mil, the abbreviation of millimeter, — one-thousandth of a liter 
— is substituted for the term cubic centimeter. 



Milton's reagent: Dissolve mercury in an equal weight of concentrated 
nitric acid; heat to solution; add two volumes of water. Decant the next day. 

Potassium ferrocyanide solution: 5 % 

Potassium hydroxide solution: 20% also 0.1% 

Potassium sulphocyanide solution: 5% 

Physiological saline solutions: 

For cold-blooded animals: 

Sodium chloride 6.5 grams 

Water 1000. mils 

For warm-blooded animals: 

Sodium chloride 9. grams 

Water 1000. mils 

Schultze's reagent: 

Anhydrous zinc chloride 25. parts 

Potassium iodide 8. parts 

Water 8.5 parts 

Iodine crystals to saturation. 

Sodium carbonate solution: 1 % 

Sodium hydroxide solution: 20% 

Stoke' s fluid: 

Ferrous sulphate 2 grams 

Tartaric acid 3 grams 

Water 100 mils 

Water of ammonia, enough to make slightly alkaline. 



GENERAL DIRECTIONS 

It is desirable that the physiologic work should be correlated so 
far as possible, i. e., the lectures, recitations and laboratory work 
should be given during the same term and have as close connection 
with each other as possible. If this is not practicable then the 
recitations or lectures should precede the work in the laboratory. 

Quizzes on the work gone over may be regarded as a part of the 
laboratory course and should occur at frequent intervals. 

Notes should be taken on the experiments as soon as they are 
performed; the manual is intended as a laboratory companion and 
the blank pages are provided for this purpose. Note taking is a 
portion of the laboratory work and the notes are to be submitted to 
the instructors for correction. 

Unexcused absences, tardiness in beginning the work and leav- 
ing without permission before the end of the period will be noted, 
and the mark or standing of those concerned will be correspondingly 
reduced at the end of the term. 

Many of the reagents which are to be frequently used will be 
placed on the student's desk on or before the periods in which they 
are needed, other reagents less frequently used will be placed on the 
general shelves and the student mast not carry these general re- 
agents to his desk. 

Any special reagent or apparatus, not included in the above, 
may be obtained when needed, by handing an order for it to one of 
the assistants. 

Before beginning his experiments, the student should inventory 
his locker and check oif on the slip, furnished for the purpose, each 
piece of apparatus that is designated. The slip is then signed by 
the student and is held by the department as a receipt. At the end 
of the term the contents of each locker are examined and compared 
with the receipt. Any articles that are missing, broken or damaged 
are charged to the student. 

Where there is any doubt as to the result of the experiment or 
any indefiniteness in the reaction consult with the assistant before 
taking up a new experiment. 



FOODS 

Any substance which may be utilized for nutritive purposes may 
be considered a food. The term proximate principles refers to or- 
ganic substances which may be broken down into simpler constitu- 
ents. Protein, fat and carbohydrates are examples. The inorganic 
substances, salts and water, are necessary in the metabolism associ- 
ated with animal and plant life and may properly be considered in 
connection with food. These substances are present in suitable pro- 
portion in milk and eggs and for young animals living upon them 
exclusively they are considered a perfect food. Eggs, although a 
perfect food for the developing bird, contain an insufficient amount 
of carbohydrate for a mammal. 

In most vegetable foods carbohydrates preponderate, while in 
animal foods, such as meat, the proteins predominate. A suitable 
diet requires that these substances should be mixed in proper pro- 
portion according to the type of animal : herbivora, omnivora or 
carnivora. In green foods there is a relatively small amount of 
nutriment and in order to provide for the large meals eaten by the 
herbivorous animals there must be a correspondingly large develop- 
ment of the alimentary tract in these animals. 

In all animals a healthy and suitable diet must contain the proper 
amount and proportion of the various chemical substances and 
should be adapted to the climate, age, and amount of work done. 
The food must contain not only the necessary amount of chemical 
substances, but these must be present in digestible form. Some 
vegetables (peas, beans, etc.) contain even more protein than meat, 
but are less nutritious because less digestible, a certain amount pass- 
ing through unused, in the feces. 

Vitamines. Experiments have shown that if an animal is fed 
upon a mixture of pure protein, fat and carbohydrate with a proper 
admixture of salts and water that, no matter how well balanced the 
ration may be, it does not thrive. A young animal fed upon such 
a diet will not grow, but if a small amount of a natural food such 
as milk is added to the above mentioned diet, it will become thrifty 
and grow normally. This indicates that some substance, about 
which at present we have little knowledge, is absolutely essential for 
the diet and quite small amounts of it are usually sufficient. If 
these little known constituents are absent from a man 's diet he reacts 
in a similar way. He becomes afflicted with the so-called ' ' deficiency 
diseases," such as scurvy, rickets and Beri-iberi. The latter is a 



disease characterized by general malnutrition and neuritis or inflam- 
mation of the nerves followed by nerve-degeneration and paralysis. 
This disease has been prevalent in Japan where it was the custom of 
the natives to eat polished rice — rice grains deprived of their outer 
layer. The disease can also be produced in birds by feeding them 
upon polished rice, and both man and bird can be rapidly cured 
by adding the discarded polishings of the rice grains. The germ or 
embryo contained in the outer layer of the grain contains the little 
known substance and it is to this substance that the term vitamine 
or accessory food has been applied. Its chemical composition is, 
at present, unknown. 

Vitamine is not confined to rice grains but is found in other 
vegetable and animal foods. The amount of vitamine varies con- 
siderably ; for example, when pigeons are fed upon polished rice, as 
much as 20 grams of meat daily must be added to prevent the occur- 
rence of Beri-beri ; whereas 3 grams of egg yolk or half a gram of 
yeast is sufficient. 

The best known of the vitamines or accessory foods are : Fat 
Soluble A — This is contained in most animal fats, and is especially 
abundant in butter and cod-liver oil. It is absent from vegetable 
fats, but is present in the green parts of vegetables. Animals cannot 
. produce it for themselves, and it is therefore necessary for nursing 
mothers to receive it in their food. 

Water Soluble B : — The condition of Beri-beri is the specific re- 
sult of the absence of this substance. Both A and B are essential 
for growth in young animals. 

Water Soluble C : — This is contained in the juices of fruits 
(especially oranges and lemons) and vegetables (especially turnips). 
The absence of this substance in the food leads to scurvy. 

Oleomargarine has now become quite a staple article of diet. It 
is made with beef fat as its chief basis. It is a good food and con- 
tains the fat-soluble accessory substance. Many of the present mar- 
garines are made mainly from vegetable oils (cotton-seed oil, etc.), 
which undergo a process of hardening or hydrogenation at a high 
temperature to render them solid at ordinary temperatures. Such 
margarines are destitute of the fat-soluble accessory, and although 
their calorific value is of the usual standard, they are of inferior 
value — especially for growing infants and children. Even if pres- 
ent in a fat which undergoes hydrogenation, the high temperature 
of the process destroys it. 



PROTEINS 

1. The proteins form the chief organic eonstiuents of the animal 
body, and occur in greater or less quantity in plants. Their com- 
position is very complex and but little is known of their structure. 
All proteins contain carbon, hydrogen, oxygen and nitrogen ; most 
of them also contain sulphur ; several, phosphorus, and some, iron. 

Proteins are almost all amorphous, non-volatile, non-diffusible, 
colorless, odorless and nearly tasteless solids. They vary in solu- 
bility. When burned or subject to dry distillation, they give off a 
disagreeable odor due to ammoniacal derivatives. Proteins are also 
distinguished by the ease with which they undergo chemical change 
under the influence of reagents, ferments, or variations in temper- 
ature. They all undergo the process of putrefaction. By boiling 
with dilute acids or alkalies, and also by the action of certain fer- 
ments, the proteins undergo hydrolysis, forming simpler compounds. 

Classification. Because of incomplete knowledge of their 
structure, an accurate classification is difficult. The simplest 
method, perhaps, is according to their source. 1. Native proteins, 
which may be isolated from the organism without loss of their prop- 
erties. 2. Derived proteins, which are obtained by the action of 
heat and reagents on native proteins. 

Another classification is according to their composition. 1. 
Simple proteins. 2. Compound proteins. 

Simple proteins are the most prominent solid constituent in 
muscle, glands, and blood serum, and to a greater or less extent in 
all tissues. The average percentage composition of simple proteins 
is: carbon, 50-55% ; hydrogen, 6.3%-7.3% ; nitrogen, 15%-18% ; 
oxygen, 21%-24% ; sulphur, 0.3 %-2.5%. Some contain phos- 
phorus, 0.85%, and a few a trace of iron. 

By the action of heat or certain reagents, soluble simple proteins 
become insoluble modifications by coagulation. A coagulated protein 
cannot return to its original condition, thereby differing from a 
precipitated protein. The temperature at which coagulation occurs, 
depends upon the nature of the protein present, the reaction of the 
solution, and the presence of neutral salts — e. g. an alkaline solution 

18 



does not coagulate on boiling, a neutral solution will do so partially, 
an acid solution completely coagulates, provided the quantity of 
neutral salts present is not too small. 

The chemical knowledge of proteins is slowly progressing and 
doubtless will become more complete as time goes on. The follow- 
ing classification should be regarded as provisional. It retains many 
of the familiar names, but attempts also to incorporate some of the 
new ideas. The classes of animal proteins are as follows (Halli- 
burton) : 



1. 


Protamines. 


6. 


Phoso-proteins. 


2. 


Histones. 


7. 


Conjugated proteins. 


3. 


Albumins. 




(a) Gluco-proteins. 


4. 


Globulins. 




(b) Nucleo-proteins. 


5. 


Sclero-proteins. 




(c) Chromo-proteins, 



Protamines. These substances are obtainable from the heads of 
the spermatozoa of certain fishes, where they occur in combination 
with nuclein. They are generally accepted as the simplest proteins 
in nature and give a typical protein reaction with the copper sul- 
phate test. On hydrolytic decomposition they yield substances of 
smaller molecular weight analogous to the peptones which are called 
protones, and then they split up into ammo-acids. Diamino-acids 
are prominent among the decomposition products. The number of 
resulting amino-acids is small as compared with other proteins and 
this is confirmatory of their simple nature. The protamines differ 
in their composition according to their source and yield their pro- 
ducts in different proportions. 

Histones. These are substances which have been separated from 
blood corpuscles; globin, the protein constituent of hemoglobin, is a 
well-marked example. They yield a larger number of amino com- 
pounds than do the protamines, but diamino-acids are still relatively 
abundant. They are coagulable by heat, soluble in dilute acids and 
can be precipitated from such solutions by ammonia. This prop- 
erty of precipitation by ammonia is possessed by no other protein 
group. 

Albumins. These are typical proteins and yield a number of 
cleavage products. The albumins enter into colloidal solution in 
water, in dilute saline solutions, and in saturated solutions of sodium 

19 



chloride and magnesium sulphate. They are precipitated by satur- 
ating their solutions with ammonium sulphate. They are coagu- 
lated usually at a temperature of 70°-73°C. Serum albumin, egg- 
albumin and lact-albumin are examples. 

Globulins. The globulins give the same general tests as albu- 
mins : they are coagulated by heat but differ from the albumins 
principally in their solublities.' In general, globulins are more 
readily salted out than albumins ; they may be precipitated and sep- 
arated from albumins by saturation with sodium chloride, mag- 
nesium sulphate or by half saturation with ammonium sulphate. 
Typical globulins are insoluble and may be precipitated by removing 
the salt (as by dialysis) which keeps them in solution. Their coag- 
ulation by heat varies considerably as to temperature. The more 
common globulins are fibrinogen and serum-globulin in blood, egg- 
globulin in the white of egg, para-myosinogen in muscle, and crys- 
tallin in the crystalline lens. Certain proteins resulting from en- 
zyme coagulation on globulins such as fibrin and 'myosin should also 
be included. The real distinction between globulins and albumins 
is that on hydrolysis globulins yield glycine and albumins do not. 

Sclero -proteins. These form a heterogeneous group of sub- 
stances formerly termed albuminoids. The prefix sclero- denotes 
the skeletal origin and often insoluble nature of the members of the 
group. The following are the principal proteins of this group : 

Collagen, the substance comprising the white fibers of con- 
nective tissue. Some regard it as the anhydride of gelatin. 
Ossein, a similar substance derived from bone. 
Gelatin. This substance is obtained by boiling collagen with 
water. It possesses the property of setting into a jelly when 
the hot water cools. On digestion, like ordinary proteins, it is 
converted into peptone4ike substances and is readily absorbed. 
It cannot altogether take the place of proteins in a diet although 
it acts as a "protein sparing" food. Animals, in whose diet the 
sole nitrogenous source is gelatin, waste rapidly. This is due 
to the absence of the tyrosin and tryptophan radicals ; if these 
be added to the diet the animals thrive better. 

Chondrin. This is a mixture of gelatin and mucoid material 
obtained by boiling cartilage. 

Elastin. This is the substance of which the yellow or elastic 

20 



fibers of connective tissue are composed. It is very insoluble. 
The sarcolemma of muscle fibers and certain basement mem- 
branes are composed of a similar substance. 

Keratin, or horny material, is the substance found in the 
surface layers of the epidermis, in hairs, nails, hoofs, and horns. 
It is very insoluble, and differs from most other proteins chiefly 
in its high percentage of the sulphur-containing amino-acid 
called cystine. Neurokeratin, a similar substance, is found in 
neuroglia and nerve fibers. 

Phospho-proteins. The principal members of this group are 
vitellin, from egg-yolk; casein, the principal protein in milk, and 
paracasein, the result of the action of rennin on casein. Phosphoric 
acid is represented in their decomposition products. They should 
not be confused with the nucleo-proteins which yield products (purdn 
and other bases) which are characteristic of nucleo-compounds. The 
phosphorus is contained within the protein molecules, and not in 
another molecular group united to the protein, as in the case of the 
nucleo-proteins. The phospho-proteins are of special value in the 
nutrition of young and embryonic animals. 

Conjugated Proteins. These are complex substances in which 
the protein molecule is united to other organic materials, which are 
also usually of a complex nature. The second constituent of the 
compound is usually termed a prosthetic group. The conjugated 
proteins may be divided into : chromo-proteins, gluco-proteins, and 
nucleo-proteins. 

Chromo-proteins are compounds of proteins with a pigment. 
Hemoglobin and its allies are examples. 

Gluco-proteins are compounds of protein with a carbohy- 
drate group. They include mucins and mucoids. 

Nucleo-proteins are compounds of protein with a complex 
organic acid called nucleic acid which contains phosphorus 
They are found in both the nuclei and cytoplasm of cells. Phys- 
ically they often simulate mucin. 



In working through the experiments, a good general rule to fol- 
low — unless otherwise directed — is not to use the whole of the 
material or solution at once but only a small portion of it so that 
other tests may be tried if necessary. 

21 



2. Preparation of Egg- Albumin Solution. Break a small hole 
in the end of a fresh egg ; carefully pour out 10 mils of the white of 
the egg into a beaker. Let the yolk remain in the shell and reserve 
for later use. Add about 200 mils of distilled water to the beaker 
containing the egg-white. Stir thoroughly with a glass rod to break 
up the membranes and thus liberate the albumin. Filter through a 
piece of muslin. Any opalescence is due to the precipitation of 
globulins. Egg-white contains about 11%-12% of egg-albumin, 
together with small quantities of globulins, grape sugar, and mineral 
matter. The white of one egg will serve for a number of students. 

A igood solution for laboratory use may also be prepared by dis- 
solving 1 gram of dry albumin in 200 mils of distilled water. 

3. Heat 5 mils of the albumin solution in a test tube to boiling. 
Notice the coagulation. Add a little nitric acid, the eoagulum may 
turn yellow but it does not dissolve. 

4. Xanthoproteic reaction. To a little of the albumin solution 
in a test tube, add some strong nitric acid ; a precipitate is formed, 
white in color, which on being boiled, turns yellow. After cooling, 
add ammonia till alkaline ; the yellow color changes to orange. With 
weak solutions there may be no precipitate at all. If only traces 
of albumin are present, the yellow 'color with the nitric acid may 
fail to appear, but the addition of ammonia gives the final test with, 
perhaps, a yellow instead of an orange color. 

5. To another portion of the solution add some of Millon's 
reagent; a white precipitate is formed, which on boiling, becomes 
brick-red in color. 

6. Piowtrowski 's reaction, (also known as the biuret reaction). 
Add excess of strong solution of potassium hydroxide and then a 
drop or two of very dilute solution of cupric sulphate, When a violet 
color results. The reaction occurs more quickly if heat is applied, 
and the color deepens. 'Make a check test by using some water in- 
stead of the albumin solution. (Peptones and albumoses give a pink 
color when only a trace of copper sulphate is used.) 

7. Acidify another portion strongly with acetic acid and add 
a few drops of 5% potassium ferrocyanide. A white precipitate is 
obtained. Peptones do not give this reaction. (Albumin is also 
precipitated by lead acetate, mercuric chloride ; picric acid ; strong 
acid, e. g., nitric; tannin; and strong alcohol). 

22 



8. Make some of the albumin solution strongly acid with acetic 
acid, add a few crystals of sodium sulphate and boil. All proteins 
except peptones are precipitated in this manner. The nitrate, after 
boiling, can be used for other tests (peptones), as the acid and 
sulphate do not decompose the solution. 

9. Indiffusibility of Albumin. Place some of the solution in a 
dialyzer. The salts (crystalloids) diffuse readily. At the next 
exercise test for chlorides by adding a little silver nitrate solution 
to a portion of the diffusate. Apply to another portion of the 
diffusate any of the preceding tests for albumin. None will be 
found. Albumin belongs to the group of colloid bodies. 

10. Globulins are proteins insoluble in water, but are soluble 
in dilute saline solutions. They are coagulated by heat and are 
precipitated by saturating their solution with magnesium sulphate 
or sodium chloride, and by the addition of an equal volume of satur- 
ated solution of ammonium sulphate. They comprise (a) serum- 
globulin of blood plasma, lactoglobulin of milk, myoglobulin, my- 
osin, and musculin of muscle ; ( b ) myosinogen of muscle, a peculiar 
protein, having properties like both albumins and globulins; (c) 
fibrinogin, differing from other globulins in being precipitated from 
its solution by an equal volume of saturated solution of sodium 
chloride and by forming fibrin when acted on by the fibrin fer- 
ment; and (d) vitellin differs from globulin in not being "salted 
out" by sodium chloride. Ovavitellin of egg-yolk and crystallin 
of the lens of the eye are vitellins. 

11. Take a small portion of the yolk in a test tube and shake 
thoroughly with Yz tube of ether ! ! for several minutes. Let 
settle. Carefully pour off the ether into evaporating dish and 
repeat several times till residue is colorless or nearly so. 

(a) Let the substance in the dish evaporate till the odor of 
ether is all gone. What is the substance? Pour a little of it in 
water and note results, also after placing a drop on a piece of 
paper. 

(b), Transfer the residue in test tube to watch glass and let 
dry thoroughly. Divide into two portions. Place portion (1) in 
a test tube, fill tube x /s full of water and shake thoroughly for five 
or ten minutes. Does the substance dissolve? Let settle, pour 
liquid through "filter" and test it for protein with the biuret test. 
Save this test and compare with next. 

23 



To the undissolved residue in the tube add a little 10% solu- 
tion of sodium chloride and shake as before. Result ? Filter and 
apply the biuret test. Result ? Conclusions ? 

12. Place portion (2) in a crucible, add a few drops of con- 
centrated HN0 3 and carefully evaporate to dryness high over the 
flame, charring as little as possible. When dry, let cool. Add a 
few drops of concentrated HN0 3 again and repeat evaporation as 
above. Now gradually lower crucible into flame and heat until 
the most of the black substance disappears. Let cool, add a little 
concentrated HO and heat to boiling. Dilute with an equal vol- 
ume of water and filter. Divide into four portions. To the first 
add some potassium ferrocyanide solution, and to the second some 
potassium sulphocyanide solution. A blue color in the former and 
a red color in the latter indicates iron. 

To the third portion add a few drops of barium chloride and 
let stand. A white ppt. indicates sulphates. 

To the fourth add a few drops of cone. HN0 3 and an equal 
volume of ammonium molybdate solution. Let stand. A yellow 
crystalline ppt. indicates phosphates. 

13. Peptones. Peptones are proteins soluble in water, but not 
coagulable by heat. They are the result of protein digestion and 
are diffusible through animal membranes. Proteoses are sub- 
stances intermediate in constitution between albumins and pep- 
tones. 

14. Peptones differ from albumins as follows : They are not 
coagulated by heat ; they are not precipitated by adding sodium 
chloride ; they are not precipitated by acids or alkalies ; they are 
not precipitated by sodium sulphate ; they are not precipitated by 
potassium ferrocyanide ; they yield a pink color with Piow- 
trowski's test instead of a violet as given for albumin. Make a 
weak solution of peptone and apply Piowtrowski's test. (See 
plate I). 

15. Like albumin the}^ are precipitated by the addition of 
tannic acid ; they are also precipitated by alcohol, but it must be 
remembered that all proteins are precipitated by alcohol, and that 
the absence of other proteins must be proved before deciding that 
the precipitate with alcohol is peptone. 

24 



II 

DERIVED PROTEINS. CONJUGATED PROTEINS. SCLERO- 

PROTEINS 

16. Acid and Alkali Albumins or Albuminates are derived 
proteins. Alkali albuminates are obtained by the action of 
alkalies on native proteins to such an extent that nitrogen and 
occasionally sulphur also are eliminated from the molecule. The 
change takes place slowly at the ordinary temperature, more 
rapidly on heating. 

Acid albuminates are obtained by digesting native proteins 
with dilute acids. 

These albuminates are insoluble in water and in neutral salt 
solution, but easily soluble in the presence of a small amount of 
either acid or alkali. The solution is not coagulated by heat. The 
albuminate is completely precipitated when the solution is neutral- 
ized. A solution in dilute acid is completely precipitated by 
saturation with ammonium sulphate or sodium chloride, while the 
solution in alkali is not precipitated by similar treatment. 

Syntonin, formed during gastric digestion, is an important 
example of acid albumin or albuminate. 

17. Albuminates. — Action of acids and alkalies on albumin. 
Take three test tubes and label them A, B, C. In each, place an 
equal amount of diluted egg-white, like that used at the last 
exercise. To A add a few drops of 0.1% solution of potassium 
hydroxide. To B add the same amount of 0.1% solution of potas- 
sium hydroxide. To C add a rather larger amount of 0.2% hydro- 
chloric acid, (6.5 mils concentrated acid to 1 liter of water). 

Put all three into a warm water bath at about the temperature 
of the body (36-40C). 

18. After fifteen minutes remove test tube A and boil. The 
protein is no longer coagulated by heat, having been converted 
into alkali albumin. After cooling, color with litmus solution, 
and neutralize with 0.2% hydrochloric acid by the contact method. 
At the neutral point a precipitate is formed, which is soluble in 
excess of either acid or alkali. Quite as delicate a test may be 
obtained without the litmus by means of the contact method. A 
distinct white precipitate appears between the two layers of fluid. 

25 



19. Next remove B. This also now contains alkali-albumin. 
Add to it a few drops of a sodium phosphate solution, color with 
litmus, and neutralize as before. Note that the alkali-albumin 
now requires more acid for its precipitation than in A, the acid 
which is first added converting the sodium phosphate into acid 
sodium phosphate. (See Plate I). 

20. Now remove C from the bath. Boil it. Again there is no 
coagulation, the protein having been converted into acid-albumin. 
After cooling, color with litmus and neutralize with 0.1% alkali. 
At the neutral point a precipitate is formed soluble in excess of 
acid or alkali. (Acid-albumin is formed more slowly than alkali- 
albumin, so that it is we'll to take plenty of time). (Plate I). 

21. Metallic albuminates. Add to separate tubes of albumin 
solution, a crystal each of copper sulphate, silver nitrate and a 
small amount of mercuric chloride. In each of the three tubes 
metallic albuminates will be precipitated. 

22. Coagulated proteins are obtained by the action of heat, 
enzymes, acids, and other reagents on native proteins, by a process 
of unknown nature, and have been found in the liver and other 
glands. Fibrin is a coagulated protein formed by the action of 
the fibrin ferment on the fibrinogen of blood plasma. 

23. Proteoses or Albumoses. Proteoses are the products of 
the hydrolysis of proteins. They are important intermediate 
products in the digestion of proteins in the animal body, are 
soluble in water, not coagulated by heat, and are precipitated by 
saturating their solutions with ammonium sulphate. 

24. Conjugated proteins on hydrolysis yield as products of 
the first splitting a simple protein and some non-protein sub- 
stances. They are subdivided, according to this non-protein 
result, as hemoglobins, glycoproteins, and nucleoproteins. 

25. Hemoglobins on hydrolysis yield a simple protein and 
hematin. Hemoglobin is the coloring agent of the blood and 
enters into combination with certain gases — for instance, carbon 
dioxide, nitrogen dioxide, and hydrocyanic acid — more readily 
than with oxygen, and the poisonous properties of these gases are 
due largely to their power of satisfying the affinities of the hemo- 
globin, and in this way rendering it incapable of taking up oxygen. 

Hemoglobin is soluble in water, in dilute solutions of albumin, 

26 



^ 




?F 



u 




acld^lbumln. alkali-albumin. peptone 



of the alkalies and their carbonates, and in sodium or ammonium 
phosphate. It is insoluble in strong alcohol, ether, and in the 
volatile and fatty oils. With the spectroscope both oxyhemoglobin 
and reduced hemoglobin show characteristic absorption bands. 
Hemoglobin crystals may be obtained, which differ in shape and 
solubility in water according to the species of animal from which 
the blood is obtained. 

26. Glycoproteins yield a substance capable of reducing an 
alkaline solution of cupric oxide. They are divided into mucins, 
mucoids, and chondroproteins. 

Mucins are secreted by mucous glands and certain mucous 
membranes. Mucin also occurs in connective tissue and in the 
umbilical cord. Mucin gives the protein color reactions, and 
forms a mucilaginous solution with water containing a little alkali. 
This solution is not coagulated by heat, but forms a precipitate 
with acetic acid insoluble in an excess of acid. 

Mucoids include colloid and ovamucoid. They occur in the 
organism and differ from mucins in physical properties and solu- 
bility, and are not precipitated by acetic acid. 

Chondroproteins yield on hydrolysis chondroitin, sulphuric 
acid, and an ethereal sulphuric acid in combination with a carbo- 
hydrate. This acid and nucleic acid have the power of forming 
with proteins a compound precipitated by acetic acid, which is 
occasionally found in the urine, and is called nucleoalbumin. 
Important chondroproteins are chondromucoid, found in cartilage, 
and amyloid, found in various organs pathologically. 

27. Nucleoproteins, on hydrolysis, yield nucleins. Three 
varieties are known, differing in hydrolytic products. 

(1). Cell-Nucleins yield a protein, ortho-phosphoric acid, and 
xanthin bases, and occur chiefly in the nuclei of cells, but also in 
the protoplasm, and may pass into the animal fluids when the cell 
is destroyed. 

(2). Pseudonucleins yield protein and ortho-phosphoric acid, 
and occur in almost all animals and vegetables. Casein of milk is a 
nucleoprOtein containing a pseudonuclein. 

( 3 ) . Nucleic acid yields ortho-phosphoric acid and xanthin bases, 
nucleoprotein containing a pseudonuclein. 

All give the protein color reactions, are soluble in water eontain- 

27 



ing a little alkali, and are precipitated from this solution by acetic 
acid. Nucleins are not decomposed by gastric juice, and are obtained 
as an insoluble residue after the artificial digestion of nucleoproteins 
with pepsin. 

28. Sclero-proteins. These are a group of proteins (albumin- 
oids) whose general properties suggest them to be anomalous simple 
proteins. They consist of a number of bodies which, in their general 
characters and elementary composition resemble proteins, but differ 
from them in many respects. They are amorphous. Some of them 
contain sulphur, and others do not. The decomposition-products 
resemble the decomposition-products of proteins. 

The principal sclero-proteins are keratin, elastin, and collagen. 

Keratin occurs in the horny portions of the skin and its appen- 
dages. Burn a paring of horn (Horse's hoof) and note the char- 
acteristic odor. Heat a paring of horn or nail 1 with strong caustic 
soda and lead acetate. Note the brown or black coloration, due to 
the formation of lead sulphide. 

Elastin occurs in connective, especially yellow elastic, tissue. 

Collagen includes ossein the chief organic constituent of bone; 
chondrogen of cartilage is a collagen mixed with a small quantity of 
other material. On boiling with water, more readily with very 
dilute acid, collagens are converted into gelatin. 

Gelatin is obtained by the prolonged boiling of connective tissues, 
for example, tendon, ligaments, bone, as well as from the substance 
collagen. Gelatin is a colorless or straw-colored solid, usually occur- 
ring in flakes or sheets, swells with water, and when heated dissolves, 
forming a clear solution, with the property of preventing the forma- 
tion of preeiptates by holding them in suspension in a finely divided 
condition ; so that they pass through filter paper. 

29. Make a watery solution of gelatin (5%) by allowing it first 
to swell up n the cold water, and then dissolving it with the aid of 
heat. It is insoluble, but swells up in about six times its volume of 
cold water. Note which of the following tests differentiate the 
gelatin from albumin. 

30. After dissolving with the aid of heat, allow a small portion 
to cool ; it gelatinizes. 

31. Apply the xanthoproteic test for proteins to some of the 
dissolved portion ; make notes of any differences as compared with 
proteins in this and in the following tests : 

28 



32. Use Milton's reagent. 

33. Try Piowtrowski 's reaction. 

34. Add some acetic acid and potassium ferrocyanide to another 
portion. Is there a precipitate ? 

35. Does it coagulate by heat ? 

36. Is it precipitated by saturation with magnesium sulphate? 

37. What is the result of the addition of tannic acid? 

38. Add picric acid (saturated solution) ; if a precipitate 
appears apply heat and note any change that may occur upon 
cooling. 

39. What is the effect of adding alcohol to the gelatin solution ? 

40. Add a little solution of mercuric chloride to the gelatin 
solution. 

41. Bone. Organic basis obtained by decalcification. Place a 
small thin dry bone in dilute hydrochloric acid (1 part of the acid 
to 8 of water) for a few days. Its mineral matter is dissolved out, 
and the bone, although retaining its original form, loses its rigidity, 
and becomes pliable, and so soft as to be capable of being cut with a 
knife. What remains is the organic matrix of ossein. The experi- 
ment in the succeeding paragraph may be carried out in a subse- 
quent exercise after the bone has become thoroughly softened. 

42. Wash the decalcified bone thoroughly with water, in which 
it is insoluble, place it in a solution of sodium carbonate and wash 
again. Boil it in water, and from it gelatin will be obtained. 
Neutralize it with sodium carbonate. The solution gelatinizes. Test 
the solution for gelatin. (30^-38) . 



Ill 
CARBOHYDRATES 



43. The term carbohydrates includes an important group of 
substances, occurring especially in plants. Starch and sugar make 
up a large proportion of the parts of plants, while cellulose forms 
the chief material from which many parts of plants are constructed. 
Carbohydrates occur to a less extent in animals, where they are 
represented chiefly by glycogen and some forms of sugars. 

29 



In elementary composition they are non-nitrogenous and the 
majority consist of CH and with the H and in the same pro- 
portion as in water, that is, 2 atoms of H to 1 atom of 0. (This 
proportion is also obtained in other substances not belonging to the 
carbohydrate group). 

Carbohydrates are indifferent bodies with a neutral reaction and 
form only loose combinations with other bodies, especially with 
bases. 

Carbohydrates are classified as monosaccharids or glucoses, 
(simple sugars); disaccharids or saccharoses; polysaccharids or 
amyloses. 

The monosaccharids (C 6 H 12 6 ) include dextrose (glucose or 
grape sugar), galactose, levulose, glycuronic acid. They cannot be 
broken down into simpler sugars. 

The disaccharids (C 12 H 22 1;L ) on taking up one molecule of water 
split and yield two simple sugars. Examples are saccharose (cane 
sugar), maltose (malt sugar), lactose (milk sugar). 

The polysaccharids (C 6 H 10 O 5 ) n do not resemble sugars. They 
have no sweet taste, and form simple sugars only after several 
reactions. Examples are starch, dextrin, animal gum, glycogen, 
and cellulose. 

44. Starch (C 6 H 10 O 5 ) is one of the most widely distributed 
substances in plants, arid it may occur in all the organs of plants, 
either (a) as a direct or indirect product of the assimilation of C0 2 
in the leaves of the plant, or (b,) as reserve material in the roots, 
seeds or shoots for the later periods of generation or vegetation. 

45. Squeeze some dry starch powder between the thumb and 
forefinger, and note the peculiar crepitation sound and feeling. 

46. Place 1 gram of starch in a mortar, rub it up with a little 
cold water, and then add 50 mils of boiling water. Transfer to an 
evaporating dish and heat for ten minutes over boiling water. Does 
the starch go into solution ? Filter and test the filtrate with a drop 
or two of the iodine solution. 

47. Add powdered dry starch to cold water. Is it soluble? 
Filter and test the filtrate with a solution of iodine. A blue color 
denotes the presence of iodide of starch. 

48. To some of the boiled portion of starch, add solution of 

30 



iodine. Heat and note any change that occurs. If not boiled too 
long another change may occur when cooled. 

49. Render some of the starch mixture alkaline (by adding slight 
excess of caustic potash. Add iodine solution. What is the result ? 

50. Acidify some of the starch mixture with dilute sulphuric 
acid, and then add iodine. What is the result ? 

51. To some of the starch mixture add some solution of tannic 
acid. Note result and then heat. 

52. Place some strong starch mixture in a dialyzer and the 
latter in distilled water. Allow it to stand for some time and test 
the water for starch. 

53. Saturate a portion of the starch mixture with crystals of 
ammonium or magnesium sulphate. Filter. Dilute the filtrate 
with an equal volume of water and add a drop or two of the iodine 
solution. Is the starch precipitated by the salt ? 

54. Glycogen (C 6 H 10 O 5 ) n is a polysaccharid found in animals 
chiefly in the liver, in the leucocytes, in all embryonic tissues, and 
in muscle. It is also found in certain forms of vegetable life, e. g., 
fungi. It is known as animal starch. It forms an opalescent solu- 
tion in water, gives a reddish-brown color with iodine. On boiling 
with acids it is converted into dextrin, then maltrose and dextrose. 
The amylolytic enzymes, by hydrolysis, produce similar changes. 
Basic lead acetate precipitates glycogen. Barfoed's reagent is not 
reduced. 

55. Dextrin. (C 6 H 10 O 5 ) is an intermediate product in the 
hydration of starch. 

56. Dissolve some dextrin, about 2%, in boiling water (100) 
mils) and cool. Add iodine solution — a reddish-brown color 
appears and disappears on heating and returns on cooling. (The 
student should take two test tubes placing the dextrin solution in 
one, and an equal volume of water in the other. Add to both an 
equal volume of iodine solution and thus compare the difference in 
color). Dextrin is made commercially by heating starch to 200° C. 

57. Saturate a solution of dextrin (56) with ammonium sul- 
phate. Note result. Filter. Dilute with an equal volume of water 
and test the filtrate for dextrin. 

58. Test a solution of dextrin (56) with Barfoed's reagent and 

31 



heat. There should be no reaction with dextrin, but a precipitate is 
given with dextrose. 

59. Test a solution of dextrin (56) with a few drops of a sol- 
ution of basic lead acetate. Is there a precipitate ? (The lead acetate 
must be basic. To insure this the solution of lead acetate may be 
boiled with litharge for ten minutes, the nitrate will be basic lead 
acetate.) 

60. Cellulose. (C 6 H 10 O 5 ) n occurs in every tissue of the higher 
plants, where it forms the walls" of cell's and the great mass of hard 
parts of wood. It is also found in the outer investment of the 
animal forms known as Tunicates. Purified absorbent cotton and 
filter paper are good examples of cellulose. Cellulose is insoluble in 
the ordinary solvents, but can be dissolved in the strong mineral 
acids, being converted into dextrin. Iodine does not stain the un- 
altered cellulose, but does so after it has been acted upon by the acid. 
Cellulose is only slightly attacked by the digestive ferments of man, 
though the herbivorous animals digest it to a greater extent. By 
the continued action of acid it is converted into glucose. 

61. Immerse a small piece of filter paper or absorbent cotton 
in a 1% solution of potassium iodide. Let dry. Immerse for an 
instant in sulphuric acid and then immediately rinse in water. If 
cellulose is present a blue color will appear. 

62. Schultze's reagent will turn cellulose blue. 

63. Immerse a strip of filter paper for a moment in concen- 
trated sulphuric acid. Then rinse it immediately in plenty of cold 
water. If the time of immersion has been correct, the paper will 
be semi-transparent after washing, and as tough, as an animal mem- 
brane. It is called vegetable parchment and can be stained blue by 
iodine. 



IV 
CARBOHYDRATES 



64. Dextrose or Glucose (Grape sugar) (C 6 H 12 6 ) exists in 
fruits and in small quantities in the blood and other fluids and 
organs. It is the form of sugar found in diabetic urine. It is 

32 



readily soluble in water. Use 100 mils of a 2% solution. Dextrose 
is made commercially by boiling starch with a dilute acid. 

65. To a portion of this solution add a little iodine solution. 
Compare with starch. Iodine does not react with a reducing sugar. 

66. Heat another portion of the solution with sulphuric acid; 
— it darkens slowly. If not successful add more dextrose and repeat. 

67. Trommer 's test. To another part of the solution add a few 
drops of a dilute solution of copper sulphate, and afterwards add 
potassium hydroxide solution in excess, that is, until the precipitate 
first formed is re-dissolved and a clear blue fluid is obtained. The 
hydrated oxide of copper precipitated from the copper sulphate is 
held in solution in presence of glucose. Heat slowly, turning the 
tube in the flame. A little below the boiling point, if the glucose 
be present, the blue color disappears and a yellow (cuprous hydrate) 
or red (cuprous oxide) precipitate is obtained. If the upper sur- 
face of the fluid has been boiled, the yellow precipitate, when it 
occurs, contrasts sharply with the deep blue-colored stratum below. 
The precipitate is first yellow, then yellowish red, and finally red. 
It is better seen in reflected than transmitted light. If no sugar be 
present, only a black color may be obtained. 

68. Fehling's solution. Keep the two solutions in separate 
bottles and mix a few mils of each (equal parts) when ready to make 
a test. A deep clear blue fluid is the result of the mixture, the 
Rochelle salt holding the cupric hydrate in solution. If kept too 
long it is apt to decompose. If in doubt as to the efficiency of the 
solution boil it, and if it remains blue it is good. 

Add some of the Fehling's solution to a portion of the glucose; 
boil; a yellowish (cuprous hydrate) or reddish (cuprous oxide) 
precipitate results. 

69. Add to a portion of the glucose solution some strong potas- 
sium hydroxide solution and then a very small amount of the sub- 
nitrate of bismuth. Boil; a black precipitate results which some- 
times forms a mirror on the walls of the test tube. This is known as 
Boettger's test. Albumin because of the sulphur present, gives a 
similar reaction and if present must be removed if a reliable sugar 
test is to be obtained. 

70. The Phenylhydrazine Test. To 5 drops of phenylhydrazine 
and 10 drops of glacial acetic acid in a test tube is added 1 mil of a 

33 



saturated solution of sodium chloride. After shaking the mixture, 
add 3 mills of 5% dextrose solution and boil the contents of the test 
tube for about two minutes. The fluid is then allowed to cool slowly 
in order that the crystals may form. The canary yellow precipitate 
may be examined in from 20 to 60 minutes under the microscope for 
the characteristic glucosazone crystals. 

The f ollowing test also gives good results, but is longer : To about 10 mils 
of the glucose solution in a test tube add 0.2 gram of phenylhydrazine hydro- 
chloride, and 0.3 gram of sodium or potassium acetate. Boil in water-bath for 
20-30 minutes; then cool the test tube by allowing cold water to run upon it 
and set it aside. A yellow crystalline precipitate is formed which is known as 
phenyl-glucosazone. Examine some of this precipitate under a low power of 
the microscope and note the needle-like and feathery crystals sometimes ar- 
ranged in the form of rosettes. Phenyl-glucosazone has a melting point of 
204°C. 

71. Conversion of starch into glucose. Boil some of the starch 
solution with a few drops of sulphuric acid until the fluid becomes 
clear and a few drops of it give no blue color with the iodine solu- 
tion. Neutralize a small portion with sodium carbonate ; test it for 
glucose. 

72. Crush a piece of compressed yeast about the size of a pea. 
Place it in a test tube and add 10 mils of the dextrose solution. 
Agitate thoroughly and transfer the mixture to a saceharometer. 
Leave in a warm place for 24 hours. If fermentation occurs bubbles 
of carbon dioxide will be found in the long arm of the saccharometer. 

73. Test a portion of the dextrose solution with Barfoed's 
reagent. Compare with Fehling's. 

74. Lactose. Milk Sugar, (O x2 K 22 lx -\-TI 2 0). This is a reduc- 
ing sugar and is found in the milk of all mammals and occasionally, 
during pregnancy, in the urine. Lactose is less soluble in water 
than dextrose and is insoluble in alcohol. With pure yeast it does 
not ferment. By the action of certain other ferments, however, it 
undergoes alcoholic fermentation, with the production at the same 
time of lactic acid, forming the drinks known as "koumiss" when 
made from mare's milk, and "kephyr" when from cow's milk. The 
ordinary souring of milk is due to the formation of lactic acid from 
the lactose by micro-organisms. Lactose must be transformed into 

34 



dextrose before it can be assimilated. If injected into the veins it 
appears in the urine. Use a 5% solution of lactose. 

75. Test a portion of the solution with Barfoed's reagent. 
Compare with the similar test for dextrose. 

76. Heat a portion of the solution carefully with sulphuric acid, 
— it chars slowly. (See 66). 

77. Add to another portion excess of potassium hydroxide and 
a few drops of copper sulphate solution and heat, — a yellow or red 
precipitae appears, (like glucose). 

78. Test another portion with Fehling's solution, — there is a 
reduction like glucose, but its reducing power is not so great as 
glucose. It requires 10 parts of lactose to reduce the amount of 
Fehling's solution that will be reduced by 7 of glucose. 

79. Apply the phenylhydrazine test and compare carefully the 
form of the crystals with those obtained in the dextrose solution. 

80. Saccharose. Cane sugar, (C^H^O^). Cane sugar is 
found in plants, not in the animal kingdom. It has no reducing 
power, but is decomposed by heating with acid into a molecule of 
dextrose and one of fructose (fruit sugar). Make a 5% solution of 
cane sugar. 

81. A portion of the solution should not reduce Fehling's solu- 
tion. (Many of the commercial sugars, however, contain sufficient 
reducing sugar to do this.) 

82. Trommer's Test. Add excess of potassium hydroxide and 
a drop of copper sulphate (it gives a clear blue fluid), and heat. 
With a pure sugar there should be no reduction. 

83. Pour strong sulphuric acid on a little dry 'cane sugar in a 
test tube. Add a few drops of water with a pipette, the whole mass 
is quickly charred. 

84. Boil a solution of cane sugar with a little sulphuric acid 
added. Neutralize the solution with a little sodium carbonate and 
test for dextrose. 

85. Apply Barfoed's, Boettger's and the phenylhydrazine tests 
to portions of the cane sugar solution and note if any reduction 
occurs. 

86. Maltose. Malt sugar, (C^H^O^+ELjO). The reducing 
power of maltose is one-third less than dextrose. Maltose can be 
easily transformed into dextrose by acids and ferments, but dex- 

35 



trose cannot be converted into maltose. Maltose must be trans- 
formed into dextrose before it can be absorbed into the blood. One 
molecule of maltose decomposes into two molecules of dextrose. Use 
a 2% solution of maltose. 

87. Apply Barfoed's test to a portion of the maltose solution 
and compare with dextrose. 

88. Apply the phenyl-hydrazine test and compare the crystals 
with those obtained in the dextrose solution. 

89. To other portions of the maltose solution apply Trommer's, 
Fehling's, and Boettger's tests respectively, and compare with 
dextrose. 



V 

FATS 

90. Fats occur in both plants and animals. They are insoluble 
in water and have a lighter specific gravity. 'They dissolve in hot 
alcohol more readily than in cold, and are easily soluble in either 
gasoline, or benzol. 

Fats are composed of three elements: carbon, hydrogen, and 
oxygen. They contain a much smaller percentage of oxygen than 
the carbohydrates, the hydrogen and oxygen not being in the pro- 
pirtion to form water. When the fats are kept at the temperature 
of superheated steam or subjected to the pancreatic enzyme — lipase, 
they take up water and are split into two compounds : glycerine, 
on the one hand, and one or more of the fatty acids, on the other. 
They may be considered, then, as made up of glycerine and fatty 
acids less water. 

This splitting up of the fat molecule is called saponification. It 
occurs when the fats become rancid. It can also be effected by boil- 
ing the fat with a caustic alkali. Here, instead of the free fatty 
acid being left, it unites with the alkali to form a salt. These 
metallic salts of a fatty acid are the soaps. The soaps of the alkalies 
are soluble in water, the potassium compound being hygroscopic and 
forming soft soap. The sodium compound forms a hard soap. 

91. Neutral fats. The neutral fats of the adipose tissue of the 

36 



body generally consist of a mixture of the neutral fats, stearin, 
pahnitin, and olein, the two former being solid at ordinary tem- 
peratures, while olein is fluid, and keeps the other two soft at 
the temperature of the body. They are lighter than water: 
Sp.gr.0.91-0.94. 

92. Try the reaction of a fresh fat, like lard or olive oil, with a 
piece of litmus paper. It is neutral ; but, if the fat has been stand- 
ing for some time and has become rancid, it may be slightly acid. 

93. Test the solubility of a few drops of olive oil in a test tube 
of water. It mixes when shaken violently, but soon separates at 
the top on standing. Add now a few drops of a soap solution and 
shake again. The liquid becomes milky and the fat does not sep- 
arate. If the oil is not fresh it may be necessary to add a few drops 
of sodium carbonate to neutralize the free acid. 

94. Take a little lard or olive oil, and observe that fat is soluble 
in ether, also chloroform. Take some of the ethereal solution of 
lard and let some of it fall upon some paper. The ether soon 
evaporates but a permanent greasy stain is left. 

95. Shake a few drops of cod-liver oil with a small amount of 
dilute solution of sodium carbonate. The mass should become white 
— an emulsion. In an emulsion the particles of oil are broken up 
into innumerable finer particles which remain discrete, that is, do 
not run together. Milk is a typical emulsion. Examine some of 
the cod-liver oil emulsion under the microscope. 

96. To albout 10 grams (11 mils) of olive oil add 20 mils of a 
10% solution of potassium hydroxide. Boil the mixture, gently 
stirring, meanwhile, until the odor of the oil has largely disappeared 
and it appears homogeneous and no oil separates when a few drops 
are poured into water. This may require half an hour. Add water 
as the solution evaporates, to keep the original volume. The product 
is a mixture of potassium soap and glycerine. 

97. Convert a portion of the above soap into the sodium or hard 
soap by adding some saturated salt solution and allowing it to stand 
until cold. It will dissolve on warming. 

98. To another portion add some solution of calcium chloride. 
A calcium soap is formed which, is insoluble in water. It is this 
compound which is produced by the action of soap on "hard water. ' ■ 

37 



Many of the heavy metals give similar compounds, Solutions of 
lead, iron, copper, etc., may be tried. 

99. To the remainder of the potassium soap solution add sul- 
phuric acid slowly until it is plainly acid to test paper. The fatty 
acids are set free as insoluble substances, the glycerine remaining 
in solution. Filter out the acids by means of a wet filter paper, 
through which the acids will not pass. The filtrate contains the 
glycerine, and must undergo still further treatment before the 
glycerine can be obtained in pure form. 



VI 

EXAMINATION OF A TEST SOLUTION FOR PROTEINS 
AND CARBOHYDRATES 

100. Note the physical characters of the solution as to color, 
transparency, odor, and taste. A persistent froth suggests an albu- 
minous solution. Filter the solution if not clear. Divide it into 
two portions, and follow the outlines below. 

A. Proteins. 

Test reaction to determine if acid or alkaline. 

Neutralize. If a precipitate forms it is acid or alkali 
albumin. If either is present, filter. 

Pour a few drops of the filtrate into water. A precipitate 
or turb,idity shows globulins, if present. 

Pour remainder of filtrate into excess of water and filter. 

To this filtrate add acetic acid and sodium sulphate and 
boil. Albumins, if present, are precipitated. If precipitate 
. is formed, filter. 

Test filtrate for gelatin. If present, saturate thoroughly 
with ammonium sulphate (crystals) and filter off preciptated 
gelatin. 

Test filtrate for peptones. Biuret test (cold). 

B. Carbohydrates. 

Test original solution for starch. 

Then saturate thoroughly with ammonium sulphate 

38 



(crystals). Starch and glycogen, if present, are precipitated 
together with proteins. Dextrin, if present, will remain in 
solution. Filter, and save precipitate (a) and filtrate (6). 

(a) Wash precipitate on filter with small portions of a 
saturated solution of ammonium sulphate till portions of 
washings give no trace of dextrin. (In testing washings for 
dextrin dilute each time with an equal volume of water). 
When washings are entirely free from dextrin pass two or 
three mils of water (cold) through filter and test for glycogen 
with a single drop of iodine. A red brown or mahogany 
color results if glycogen is present. Basic lead acetate pre- 
cipitates glycogen but not dextrin. 

(b) Dilute filtrate (b) with an equal volume of water 
and test for dextrin. 

Test original solution for reducing sugars — first precipitating 
out the proteins with acetic acid and sodium sulphate and boiling. 



VII 

SALIVARY DIGESTION 

101. The saliva is a mixture of the secretions of the parotid, 
submaxillary, and sublingual glands with that of the glands of the 
membrane of the mouth. The reaction of the mixed saliva is usually 
alkaline but may on fasting, also during the night toward morning, 
and 2-3 hours after meals, or after much talking, become acid. 
On standing some hours it may 'become acid and a film of calcium 
carbonate form on the surface. The normal mixed saliva contains 
inorganic constituents consisting of: carbonates, chlorides, sul- 
phates, and nitrites of magnesium, calcium, potassium, and sodium, 
also the sulphocyanide of potassium. The nitrites and sulpho- 
cyanide are often absent. The organic constituents are albumin, 
mucin, and ptyalin. The ptyalin has the power to convert starch 
into dextrin, maltose, and some dextrose. It is not able to penetrate 
the granule of unboiled starch, or does so very slowly, differing in 
this respect from the corresponding enzyme of the pancreas. It 

39 



acts best at about the temperature 40° C. Ptyalin is destroyed by 
acids — especially, the mineral acids. In the saliva of some animals, 
as the horse and dog, the enzyme is weak or absent. 

To obtain mixed saliva. Chew a small piece of paraffin or 
chewing gum, or inhale ether for a short time to stimulate the flow 
of the secretion. Collect it in a graduate until you have about 50 
mils. Note that, in a short time, more or less of a sediment occurs 
due to the deposition of epithelial cells, d'ebris of food, bacteria, etc. 
Numerous air bubbles are usually present upon the surface. 

Filter. Is it translucent? Is there any great amount of 
viscidity? What is its reaction to litmus paper? The specific 
gravity is 1002-1006. Test the specific gravity with an urinometer. 

102. To a small portion add acetic acid. A precipitate indi- 
cates mucin. Not soluble in excess. 

103. With another portion test for traces of proteins with the 
xanthoproteic reaction and Millon's test. 

104. To a few drops of saliva in a porcelain evaporating dish 
add a few drops of dilute acidulated ferric chloride, — a red colora- 
tion indicates the presence of sulphocyanide of potassium, the color 
does not disappear on heating, nor on the addition of an acid, but is 
discharged by mercuric chloride. Meconic acid gives a similar color, 
but it is not discharged by mercuric chloride. The sulphocyanide 
is present only in the secretion from the parotid gland. 

105. Test for nitrites with a few drops of a starch solution 
acidified with a little dilute sulphuric acid and containing a small 
amount of potassium iodide. A nitrite immediately gives a blue 
color. 

106. Test for chlorides by adding to the saliva a few drops of 
nitric acid followed by a few drops of silver nitrate. A white 
precipitate indicates the combination of the chloride with the silver 
to form silver chloride. 

107. Test another portion of the saliva with a few drops of 
barium chloride for sulphates. 

108. Digestive action on starch. Prepare a mixture by placing 
1 gram of starch in a mortar and adding a few mils of cold water, 
and mix well with the starch. Add 200 mils of boiling water, stir- 
ring all the while. Boil the fluid for a few minutes. This gives a 
0.5% mixture. 

40 



109. Dilute the saliva wrbh an equal volume of distilled water. 
Label four test tubes, A, B, C, and D. Into A place some saliva, 
boil it and later add some starch mucilage. In B and C, place 
starch mucilage, to B add a few drops of concentrated hydrochloric 
acid and to C some 20% potassium hydroxide, and add saliva to 
each tube. To D add merely the saliva to the starch mixture. 

Place all four in a water bath not exceeding 40° C, and after a 
time test a small portion of them for sugar with Fehling's solution. 
Reserve a small amount of D. Why is no sugar formed in A? 
In B and C a strong acid and alkali arrest the action of ptyalin. 
Neutralize a portion of B and C and test again. Is there any result ? 
In D the starch has been converted by the ptyalin into a reducing 
sugar. 

110. Test portions of D with Fehling's and iodine solutions. 
The absence of any blue color with the iodine indicates that the 
starch has disappeared having been converted into a reducing sugar 
— maltose. Also test the remainder of A, B, and C with the iodine 
solution. 

111. Test another portion of D with phenylhydrazin, (70) 
crystals of phenyl-maltosazone should develop. Examine under the 
microscope. 

112. The intermediate products of salivary digestion may be 
detected by proceeding as in D (109) and testing a few drops of 
the mixture every two minutes with a drop of iodine upon a porce- 
lain plate. At first there is a blue color denoting soluble starch ; 
later there is a reddish violet color indicating the presence of 
erythrodextrin; still later there is only a slight yellowish brown 
color, or no color at all, when the drop of iodine is added, and this 
indicates achroodextrin — the achromic point — when a reducing 
sugar maltose is also present. At this point the solution should 
reduce Fehlings. Any undigested starch may be precipitated by 
alcohol which leaves the maltose in solution. Saturation with am- 
monium sulphate crystals also precipitates the starch but does not 
affect the dextrins or maltose. 

113. The effect of drugs on salivary action. The following may 
be used; carbolic acid 2%, saturated aqueous solutions of benzoic 
and boric acids, alcohol 50%. Place in each test tube 2 mils of boiled 
starch 2%, 2 mils sodium carbonate 1%, 5 mils of the given drug, 

41 



and 1 mil of saliva. Shake and set in the water bath at 40°C. for an 
hour. The activity of digestion may be compared by testing a 
small portion of each tube with the iodine solution, to see if the 
starch has disappeared ; or another small portion with Fehling's to 
see if maltose has formed. A cruder method is to add to each tube 
an equal volume of 10% caustic soda or potash with a little dilute 
copper sulphate solution (Trommer's test). The amount of pre- 
cipitate or depth of color roughly corresponds to the amount of 
digestion. 

114. Bread. Crumble up a small piece of bread in a test tube 
and add some cold distilled water until it softens and with slight 
shaking disintegrates. Divide the mixture into two portions. 

115. Apply a drop of iodine solution: b,lue color indicates 
starch. 

116. Apply the xanthoproteic reaction to the other portion. 
Any crumbs that may be in the solution if colored orange would 
indicate the presence of a protein. The liquid portion may not 
show as cLeep a color, indicating a lesser amount in solution. 

117. Try the above tests hurriedly by dropping a little iodine 
solution upon the bread. Similarly with the xanthoproteic test by 
letting a drop of nitric acid fall upon the Ibread and then a drop of 
ammonia upon the spot already covered by the nitric acid. 

118. Potato. Boil a small piece of potato in water and let it 
cool. Divide the liquid into two parts. 

Test one portion for starch with the iodine solution. Without 
boiling, the starch might give no reaction as the granules are en- 
closed in a coating of cellulose. 

119. Apply the xanthoproteic test. Only a faint orange color 
appears, indicating that very little protein is present. 

120. Small portions of ground' oats, corn, wheat, or bran may 
be mixed in separate tubes with saliva and digested. The inter- 
mediate and end products may be tested for as in 112. 



42 



VIII 
GASTRIC DIGESTION 

121. The gastric juice, secreted by the glands of the stomach, 
d liters from the other digestive fluids in having an acid reaction. 
It is a clear thin liquid having a specific gravity of 1002-1006. 
The average composition of man 's gastric juice is as follows : 

Water 99.26 

Pepsin, rennin and other organic matter 0.30 

Free hydrochloric acid 0.22 

Alkali chlorides 0.20 

Phosphates of alkalies, calcium, magnesium and iron 0.02 

There is more hydrochloric acid than can unite with the bases, 
and' this must consequently be in the free state. The most im- 
portant of the organic substances are the enzymes: pepsin and 
rennin. Differences exist in different animals, e. g., in carnivora 
there is a higher percentage of acid than in others. 

An artificial digestive fluid giving very good results may be made 
by dissolving 0.3 gram of commercial pepsin in 1000 mils of a 0.2% 
solution of hydrochloric acid. 

It is more desirable in many ways, however, to prepare an extract 
from the gastric mucous membrane itself. The writer has found 
the following method to answer very atisfactorily. To each gram 
of the mucous membrane add 1 mil of a 1% solution of acetic acid. 
Triturate thoroughly in the mortar ; then add 10 mils of chloroform 
water for each gram of the mucous membrane. This may be kept 
for some time. When ready for use, filter and add 2 mils of the 
extract to 8 mils of the 0.2% hydrochloric acid, or equal volumes of 
the extract and the acid may be used. 

From a comparative standpoint, extracts may be made from each 
of the three great groups of animals: omnivora (pig), carnivora 
(dog), and herbivora (horse or cow), and differences in the rate of 
digestion noted. 

122, Label six test tubes, A, B, C, D, E, F. In A fill the tube 
half full of distilled water, and add 30 drops, or 2 mils of the 
extract. Fill B half full of 0.2% hydrochloric acid. Treat C simi- 

43 



larly to B, but add 30 drops of the extract. Fill D half full of a 1% 
solution of sodium carbonate and add 30 drops of the extract. Place 
30 drops of the extract in E and add 2 or 3 mils of distilled water 
and boil, then add enough 0.2% hydrochloric acid to make the tube 
half full. In F put 2 mils of the extract and 2 mils of bile and fill 
the tube half full with 0.2% hydrochloric acid. In each of the 6 
test tubes put a small thread of well-washed and boiled fibrin. Place 
all the tubes in a water bath at 40°C, and after an hour note any 
changes that may have occurred in any of the tubes. The rapidity 
of action will indicate the strength of the enzyme. Explain in your 
notes why no action has occurred in certain of the tubes. Test tube 
C is to be plugged with cotton and reserved for later examination, 
in the next exercise. 

123. The following tubes are to be prepared exactly as C but 
omitting the fibrin. In the first tube a very small piece of meat ; in 
the second a crumb of bread ; in the third a bit of boiled potato ; in 
the fourth add a small piece of butter ; in the fifth, 1 mil of milk 
diluted with 5 mils of distilled water ; in the sixth test tube a small 
piece of gelatin. These tubes, also, are to remain in the water bath 
at 40 °C, and tested later for intermediate and end products. (See 
125, 126, 128). 



IX 
GASTRIC DIGESTION 

124. The contents of tu'be € are to be divided into 4 parts, 3 of 
which are to be used in the following tests, and the other part to be 
held in reserve, if needed, to correct any of the other tests. 

125. Color one portion of the fluid with the litmus solution and 
neutralize by the contact method with 1% sodium carbonate. (See 
plate I). At the neutral zone a precipitate will appear indicating 
acid-albumin (syntonin). The contact test without the litmus is 
equally delicate. 

126. Add to another portion of the solution enough crystals of 
neutral ammonium sulphate to saturate it. This brings down the 

44 



proteoses or albumoses in the form of a white precipitate. Proteose 
like peptone is soluble in water, and gives the biuret reaction. Am- 
monium sulphate precipitates all of the proteins but peptone. 

Another test for proteose is to add sodium chloride and a few drops of 
nitric acid. A precipitate should aippear which is dissolved on heating, but 
reappears on cooling, indicating the presence of proteose. 

127. Peptones behave differently from the native proteins in 
the copper sulphate and potassium hydroxide test, if only a trace 
of copper sulphate is used. They give a pink instead of a violet 
color. (Also true of proteoses ) . The pink color is also given by the 
substance called biuret, hence the test is often called the biuret 
reaction. (Biuret is formed by heating urea; ammonia passes off 
and leaves biuret, thus : 2CON 2 H 4 (urea) — NH 3 (ammonia) equals 
C 2 2 N 3 H 5 ('biuret). 

128. To the third portion add neutral ammonium sulphate to 
saturation. This precipitates all of the proteoses and proteins while 
the peptones remain in solution. Filter and test the filtrate for 
peptones -by the biuret test as follows : Take another test tube and 
put a few drops of 1% solution of copper sulphate in it; empty it 
out so that the merest trace of the copper sulphate is adherent to 
the wall of the tube, then add the filtrate and a few drops of strong 
potassium hydroxide. A pink color (biuret reaction) should be 
produced. 

129. If digestion has been quite long and complete the tests for 
acid-albumin and proteose may not be very satisfactory as these 
substances may have been converted into peptones. They are more 
readily found shortly after digestion has begun. The main fact, 
however, that an indiffusible protein, before 'being converted into 
a diffusible peptone, must pass through intermediate forms — acid 
albumin and proteose — is important, and must be kept in mind in 
this and succeeding experiments. 

130. After filtering, treat the contents of the tubes containing 
meat, bread, potato, butter, milk, and gelatin, for acid albumin, 
proteose and peptone. 

131. Drugs on gastric digestion. Use the same preparations 
as in 113. Put in each test tube 4 mils of 0.2% hydrochloric acid, 

45 



2 mils of gastric extract and 4 mils of the given drug. Keep the 
tubes at 40° C. for a number of hours or over night and test for 
peptones. 

132. Take two pieces of moist fibrin of equal size. Tie one of 
the pieces firmly in a bunch with thread and place it in a test tube 
containing some gastric extract and 0.2% hydrochloric acid. Tear 
the other piece of fibrin into small flakes and place it in another test 
tube with the same amount of extract and acid. Let the two tubes 
digest at 40° C. for an equal length of time and note in which most 
digestion has occurred. In a crude way this experiment shows the 
effect of mastication upon gastric digestion. Large lumps are acted 
upon slowly and with difficulty, while an equal amount of material 
in a state of fine division is readily digested. 

133. Rennin or chymosin is the milk-curdling enzyme of the 
stomach. It is apparently a constant constituent of the gastric 
juice of vertebrates. It is especially abundant in the mucous mem- 
brane of the stomach of the calf (rennet). 

A solution for experimental purposes may 'be prepared as in 121. 
Both rennin and pepsin go into solution. The preparation should 
not stand too long (2 or 3 days), and should be neutralized with 
1% sodium carbonate before using. Pepsin digests rennin in an 
acid medium. Commercial rennin may be used in fluid or tablet 
form experimentally. 

134. To 10 mils of milk in a test tube add a few drops of the 
fluid extract of rennin or a 1 grain tablet of rennin and keep the 
tube for some minutes at a temperature of about 38 °C. After a 
short time the milk becomes solid, forming a curd, and after a time 
the curd of paracasein contracts and squeezes out a fluid — the whey. 

135. Repeat the experiment but first boil the rennin. Compare 
and explain the result. 

136. Half fill a test tube with 0.2% hydrochloric acid. Put in a 
little fibrin and add a tablet of rennin. Keep at a temperature of 
38°-40°C. for a few hours and test for peptones and intermediate 
products. 

137. To 10 mils of milk in a test tube add a few flakes of com- 
mercial pepsin. Keep at a temperature of 38 °C. and note if there 
is any coagulation of the milk. See that the reaction is neutral. 
Test also for peptones. 

46 



138. Digest with the gastric extract in separate tubes small 
amounts of ground oats, corn, wheat and bran, as in sections 
123-128. 

139. Gastric lipase. After the protein envelopes of the fat cells 
are digested, the solid fats are liquified. They are then split to a 
small extent into their constituents : glycerin and fatty acids. This 
action may be brought about by a regurgitation of the contents of 
the duodenum mixed with the pancreatic juice. Even after regurgi- 
tation has been prevented by ligating tbe pylorus a small amount of 
fat cleavage occurs and this indicates the presence of lipase in the 
gastric juice. It is said that the administration of fat in the food 
increases the regurgitation from the duodenum. 



X 

PANCREATIC DIGESTION 

140. The pancreatic secretion is a clear thick alkaline fluid, 
rich in solids, and possesses very 'active enzyme properties. It con- 
tains at least three distinct enzymes, besides albumin, leucin, fats, 
soaps and salts. These solid constituents make up about 10'% of 
the secretion. The enzymes occur in the gland in the form of 
inactive zymogens, but are changed to the active form after being 
discharged. The reaction of the juice is alkaline from the presence 
of sodium carbonate. The extract made from the gland by means 
of warm water may be acid in reaction from the presence of sarco- 
lactic acid, especially if the gland is extracted some time after death. 

The ingestion of food has more or less influence upon the flow of 
the pancreatic fluid. There is, therefore, no secretion during starv- 
ation and it is intermittent in carnivorous animals where some time 
elapses between meals. On the other hand secretion is going on 
almost continually in herbivorous animals because digestion is almost 
uninterruptedly taking place. 

The enzymes found in the pancreatic juice are : trypsin which 
digests proteins in an alkaline medium, amylase which digests starch 
similarly to ptyalin, lipase which splits up fats into glycerine and 

47 



fatty acids, and finally there is some evidence of a milk curdling 
enzyme, although the latter is not universally accepted. 

A substance, secretin, derived from prosecretin in the duodenal 
mucosa in conduction with the presence of hydrochloric acid, has the 
power of stimulating the flow of the pancreatic juice. It is believed 
that secretin liberates amylase and lipase from their precursors 
proamylase and prolipase and these two active enzymes pass into 
the pancreatic juice. It also liberates trypsinogen from its pre- 
cursor protrypsinogen setting it free in the pancreatic secretion, 
•but the trypsinogen is itself inactive until brought in contact with 
another enzyme — enterokinase — produced in the intestinal mucosa. 
Contact of the trypsinogen with enterokinase produces trypsin, 
which has an intense action upon protein material. The pancreatic 
juice is therefore inactive so far as protein digestion is concerned 
until it has come in contact with the intestinal mucosa. The idea 
has been advanced that trypsinogen is a complex consisting of 
trypsin combined with a protein and that as long as this combined 
form exists it is inactive ; the enterokinase is believed to digest this 
protein thus liberating the trypsin into its active form. 

A pancreatic extract for dligestive purposes with trypsin and 
amylase may be made by running the gland through a food chopper, 
or triturating it to a pulp in a mortar and adding 1 mil of 
1% acetic acid for each gram of the pancreas. Then add 10 mils 
of chloroform water for each gram of the pancreas to extract the 
enzymes and at the same time, on account of its antiseptic properties, 
to prevent putrefaction. For use, 2 mils of this extract may be 
added to 8 mils of 1% sodium carbonate, or equal volumes of the 
two may be employed. 

Commercial pancreatin 5 grams dissolved in 200 mils of 1% 
sodium carbonate will also serve for experimental purposes. 

141. Prepare seven test tubes. Each test tube is to be half 
filled with 1% sodium carbonate and 2 mils of the pancreatic extract 
added. To tubes prepared as above add the following : 1, a bit of 
fibrin ; 2, a piece of meat ; 3, a crumb of bread ; 4, a bit of cooked 
potato; 5, a bit of cheese; 6, a small piece of gelatin; 7, a small 
amount of each of the above substances. Keep these in the water 
bath at 40° C. Note particularly any changes that may occur in No. 
1, and compare with the fibrin digested with the gastric juice. In 

48 



those tubes which first show signs of digestive action, test the con- 
tents for alkali-albumin by neutralization. ('Similar to the acid- 
albumin test the only difference being the reaction of the digestive 
fluid). Test also for proteoses. Place the tubes in the incubator 
until the next exercise and, after filtering, again test them for alkali- 
albumin, proteose and peptone, as with preceding tests. (125-128) . 
Reserve a portion of the contents of tubes No. 1 and 2 for indol test 
later. (152). 

142. Place a bit of fibrin in two test tubes. Half fill the tubes 
with 1% sodium carbonate. To one tube add 2 mils of the pan- 
creatic extract. To the other tube add 2 mils of bile and 2 mils of 
the pancreatic extract. Let the tubes digest at 40° C. and note in 
which tube peptone first appears. 

143. Take three test tubes, add 5 mils of boiled starch mixture 
and 5 mils of 1% sodium carbonate to each tube. To No. 1, add 
2 mils of the pancreatic extract. To No. 2, add 2 mils of bile. To 
No. 3, add 2 mils of bile and 2 mils of the pancreatic extract. Place 
the three test tubes in the water bath at 40° C. Test a few drops 
from each tube every minute upon a white plate with a drop or 
two of iodine and note in which tube the starch first disappears. 

144. Prepare two tubes for starch digestion as follows : In one 
place 5 mils of 2% boiled starch and 5 mils of 1% sodium carbonate 
and 2 mils of pancreatic extract. In the other place 1 gram of 
unboiled starch triturated in 5 mils of cold water, add 5 mils of 1% 
sodium carbonate and 2 mils of pancreatic extract. Place both 
tubes in the water bath at 40°C. and test at intervals as in No. 143. 
Continue the digestion long enough to find sugar in both tubes by 
the Fehling's test. 



XI 

PANCREATIC DIGESTION 

145. Prepare four test tubes as follows, labeling them in order, 
1, 2, 3, 4, and adding a bit of fibrin to each tube. To No. 1 add 30 
drops of the pancreatic extract from the pig, and some distilled 

49 



water ; to No. 2, the same amount of the pig's pancreatic extract and 
excess of 0.2% hydrochloric acid; to No. 3, some 1% sodium car- 
bonate alone ; for No. 4, put 30 drops of the pancreatic extract into 
a separate test tube, add a little of the 1% sodium carbonate and 
boil, and then add the fibrin. Put all of the test tubes in the water 
bath at 40° C. After a few hours examine them and explain the 
results. 

146. Emulsion Experiment. Shake up a few drops of olive oil 
in a test tube with 2 mils artificial pancreatic juice and 2 mils 1% 
sodium carbonate. Place the mixture for a few minutes in the bath 
at 40° C, and shake again, compare the results before and after 
warming. If the oil is neutral, there may be no emulsion or only 
a poor one. The addition of a few drops of oleic acid will improve 
it. Why? 

147. In another tube with a little olive oil add 2 mils of bile and 
2 mils of 1% sodium carbonate, shake and place in water bath at 
40° C. and compare the emulsive effect with 146. Note whether the 
oil, is neutral or not. 

148. Action on Fat. For this experiment it is necessary that 
the fat should be perfectly neutral. Commercial oils usually contain 
free fatty acids. 

The following method has been recommended for neutralization by Kruken- 
berg : Place the oil in a porcelain capsule and mix it with not too much baryta 
siolution, (baryta mixture is prepared by mixing one volume of a solution of 
barium nitrate and two volumes of barium hydrate, both saturated in the cold), 
and boil for some time. Allow it to cool. The unsaponified oil is extracted 
with either. The ethereal extract is separated from the insoluble portion and 
the ether evaporated over warm Water. (The flame must not be brought near 
the ether. Let the water come to a boil, put out the flame and then put the 
dish containing the ether upon the hot water). The oil should now be neutra- 
lized. 

The cream from fresh milk is usually of a neutral reaction and 
serves very well in the following experiments. 

149. Take two test tubes and place in each 2 mils of cream — 
neutral fat. Add 1 mil blue litmus to color. In the first tube place 
a small piece of fresh pancreas. Put both tubes in the water bath 
and observe at intervals. Note if any change of color occurs in the 
one with the pancreas, due to the formation of fatty acids by the 

50 



enzyme lipase. A fresh watery extract of the pancreas also acts 
favorably. 

Another form of the experiment is to mix the oil with finely divided per- 
fectly fresh pancreas in a. mortar, and keep it for a time at 40 °C. It soon 
becomes acid, owing to the formation of fatty acids. Test with litmus paper. 

150. Action on milk. Dilute 2 mils of cow's milk with 10 mils 
of distilled water in a test tube and 'add 5-6 drops of pancreatic 
extract. Keep at 40° C. from y 2 to 1 hour. Note any change that 
has occurred. 

151. Divide the above into two parts. To one part add a little 
dilute acetic acid; if there is no precipitate it indicates that the 
casein has been converted into peptones. To the other part apply 
the biuret reaction for peptones. 

152. With the reserved portion from the albumin and fibrin 
tubes, (141), indol may be found if digestion has continued long 
enough and if an offensive odor be present. To some of the sus- 
pected fluid add 1 cc. of 0.01% solution of sodium or potassium 
nitrite ( fresh ) and then a few drops of concentrated sulphuric acid. 
A pink color indicates the presence of indol. 

153. 'The action of trypsin is similar to that of pepsin but goes 
further and is effective only in an alkaline medium. Trypsin 
rapidly splits up the proteose and peptone which have resulted from 
peptic digestion into simpler substances, the polypeptids. These in 
turn are broken up into their constituents, the amino-acids, such as 
leucin, tyrosin, alanine, aspartic acid, glutamic acid, arginine, 
tryptophan, and others. In addition to these there is a certain 
amount of ammonia. The presence of tryptophan is indicated by 
the red color which a tryptic digest gives with chlorine water or 
bromine water. 

154. The action of the pancreatic juice in its conversion of 
starch into maltose is the most rapid. Its power is greater than 
that of the saliva and it will act to a certain extent even upon 
unboiled starch. Amylase is absent for a time in the pancreatic 
juice of infants and its absence indicates that starch is not a suitable 
food in their early days. 

155. If a glycerin extract of the pancreas be filtered, the filtrate 
has no fat-splitting action, neither has the residue left upon the 

51 



filter ; but if the residue and filtrate be mixed together a strong fat- 
splitting action is obtained. Lipase is separable into two portions. 
A substance in the residue represents an inactive lipase ; while 
another substance in the filtrate represents its co-enzyme. The co- 
enzyme is not destroyed by boiling. Bile salts also activate the 
inactive lipase and this fact explains why the presence of bile favors 
the splitting of fat. 

156. Succus Entericus. The intestinal juice contains inverting 
enzymes Which convert disaceharids into monosaccharids. Invertase 

converts cane sugar into dextrose and levulose (fruit sugar) ; 
maltase converts maltose into dextrose, and lactase converts lactose 
into dextrose and galactose. Invertase is also found in yeast cells. 
The succus entericus also contains another enzyme called erepsin. 
This is a proteolytic enzyme and breaks up the proteoses and pep- 
tones into their final products, the amino-acids, assisting in this way 
the action of trypsin. The only native protein which erepsin digests 
is casein. 

An extract may be made from the small intestine by adding to 
each gram of the mucous membrane used 1 mil of 1% acetic acid, 
triturating in the mortar and adding 10 mils of chloroform water 
for each gram of the mucous membrane. Let the mixture stand for 
a day or two and before using make slightly alkaline by the addition 
of 1% sodium carbonate. 

157. Place 10 mils of a 10% solution of cane sugar in a test tube 
and add 2 mils of the extract of succus entericus. Allow the mixture 
to digest at 40° C. After a short time test with Fehling's solution 
for reducing sugar. 

158. From the mucosa of the intestine there is also obtained 
enterokinase an enzyme which activates the inert trypsinogen, con- 
verting it into active trypsin. Take two test tubes and place in each 
the same amount of fibrin. Add the usual amount of pancreatic 
extract and sodium carbonate solution to both tubes. To one of the 
tubes add a few drops of the intestinal extract allowing the other 
to remain as it is. Place both tubes in the water bath and observe 
in which tryptic digestion first occurs by making occasional tests for 
peptones. 

In the ordinary preparation of pancreatic extracts there is 
usually more or less contamination with the intestinal mucosa so 

52 



that in this way there may be introduced into the pancreatic 
extract a small amount of the enterokinase sufficient to activate the 
trypsinogen. 

159. Test the action of the pancreatic extract with 1% solution 
carbonate upon some ground oats, corn, wheat, and bran in separate 
tubes. See if results are obtainable from tryptic and amylolytic 
digestion. 



XII 
BILE 

160. Bile is a mixture of the secretion of liver cells and of mucin 
derived from the cells lining the gall bladder and duct. The bile 
obtained directly from the liver contains about 2%, and that from 
the gall bladder contains about 12% of solids. The difference is 
due to concentration in the gall bladder and ducts, where also 
mucinous substances are added. The bile is normally a reddish 
brown or grenish viscid fluid with a bitter taste and a neutral or 
slightly alkaline reaction. After a protein diet the secretion is 
increased, whereas with fats and carbohydrates it is less marked. 
The secretion is also decreased in starvation. 

The compounds which make up the larger part of the solid 
matter of the bile are the sodium salts of glycocholic and tauro- 
cholic acids. Besides these and the b,iliary mucin there are present 
fats, soaps, lecithin, and cholesterin, also a nmber of inorganic salts 
of the alkalies, alkaline earths, and iron. The color of the bile is 
due to the biliary pigments, bilirubin, biliverdin. The source of 
bilirubin is undoubtedly hematin. On reduction it yields hydro- 
bi'lirubin which is closely related if not identical with stercobilin 
(found in the intestines, giving color to feces), and with urobilin of 
urine. On oxidation bilirubin yields biliverdin. The amount of 
pigment in the bile is usually only a few hundredths of a per cent., 
rarely 0.1%. As to the origin of these bile constituents it may be 
said that the bile acids are elaborated by the cells of the liver, not 
elsewhere in the body. The bile pigments may possibly be formed 
in other parts of the body than in the liver, but under normal cou- 

53 



ditions the liver is the organ where they are formed. Taurin and 
glycocoll result from the decomposition of proteins in any part of 
the body. The bile contains no proteins nor formed elements, but 
in some animals a small amount of diastatic enzyme may be found. 
Secretin stimulates the production of bile as well as the pancreatic 
juice. 

161. Note the peculiar odor of bile. Pour a little from one 
vessel to another and note the viscidity, due to the presence of mucin 
and nucleo-protein. 

162. Place some dilute bile (1 to 5) in a test tube and heat to 
boiling. Immerse a strip of red litmus paper, then remove and wash 
with water. The reaction should be alkaline if the bile is fresh. 

163. To 5 mils of b,ile in a test tube add 10 mils water and then 
some strong alcohol. This produces a pecipitate of mucin with some 
pigment entangled. 

164. Mucin is also precipitated by the addition of acetic acid to 
bile. Perform this test using the same proportions as in 163. Filter 
off the mucin. 

165. To a portion of the filtrate add a little hydrochloric acid 
and potassium ferrocyanide. A blue color indicates the presence of 
iron. The experiment may be modified by placing some thin sec- 
tions of liver in a solution of potassium ferrocyanide for a few 
minutes and then in dilute hydrochloric acid. The sections turn 
bluish from the formation of prussian blue. With the microscope 
blue granules may be seen in some of the hepatic cells. 

166. Test another portion of the filtrate for proteins, also for 
chlorides and sulphates. Fresh human bile gives no spectrum, but 
the bile of the ox, mouse and some other animals does. 

167. Pettenkofer's Test for Bile Acids. Take 2 mils of clear 
diluted bile in a test tube arid add 4 drops of a 10% solution of 
cane sugar. Add strong sulphuric acid, drop by drop, cooling the 
tube in a dish of cold water immediately ofter adding the acid. Not 
more than 2 mils of the acid should be used. Too much heat causes 
carbonization of the sugar and the test is ruined. If bile acids are 
present, the fluid at first becomes opaque, then clear, and successively 
brown, red and purple. It may require an hour or more to accom- 
plish this test. This reaction depends upon the production of 
furfurol (C 4 H 3 OCHO) by the destruction of the sugar when the 

54 



sulphuric acid is added. Furfurol in turn combines with cholalic 
acid, formed by the action of the sulphuric acid on the biile acids, 
giving the color. Some other substances, as morphine, albumin, etc., 
give a very similar color, and the test must, therefore, be used with 
caution. In very dilute solutions of bile the reaction does not 
appear and cannot be used satisfactorily in testing urine for the 
presence of bile. 

Pettenkof er 's test may also be quite satisfactorily performed 
more quickly by putting a little of the bile in a porcelain capsule, 
adding a drop or two of a solution of cane sugar and then a few 
drops of strong sulphuric acid. 

168 Grmelin's Test for Bile Pigments. Ox gall does not yield 
this test as readily as that from the omnivora or carnivora. To a 
small quantity of bile, in a test-tube, add, drop by drop, nitric acid 
yellow with nitrous acid, (if the acid is clear, add a single crystal 
of cane sugar, warm, and the acid becomes yellow from the develop- 
ment of a small amount of nitrous acid, shaking after each drop ; 
the yellowish green color becomes first a dark green, then blue, then 
violet, then red, and finally a dirty yellow. The blue and violet 
colors are less obvious than the rest. 

Repeat the test in the following way : place a drop of bile in a 
porcelain evaporating dish, and place a drop of yellow nitric acid 
so that it runs into the drop of bile ; where the fluids mingle, zones 
of color, green, blue, violet, red and yellow, from the bile to the 
acid, are seen. 

169. Surface Tension Test. (Hay). In a test tube containing 
some dilute (1-2) bile, sprinkle some powdered sulphur. Repeat 
the experiment upon a test tube containing ordinary water. Com- 
pare the two and note in which the sulphur sinks the most readily. 

170. Place some diluted bile in a test tube, incline the tube and 
add cautiously 2-3 mils of a dilute tincture of iodine so that it forms 
a layer. In a short time a bright green ring forms at the zone of 
contact. After the ingestion of antipyrin, the urine will give a 
similar green ring with iodine. 

171. Acidulate some dilute bile with acetic acid, add a few mils 
of chloroform and shake. The chloroform dissolves the bilirubin 
(not biliverdin) and is colored yellow. 

172. Add a little bile to some starch mucilage as in salivary 
digestion. Test for any reducing sugar. 

55 



173. To 5 mils of undiluted bile add an equal volume of water 
and s6me alcohol to precipitate the mucin. Filter and divide the 
filtrate into two portions: to one portion add some hydrochloric 
acid which causes a precipitation of glycocholic acid; to the other 
portion add a little of a 1% solution of neutral lead acetate which 
throws down lead glycocholate. Remove this by filtration, and to 
the filtrate add a little 1% solution of basic lead acetate, which gives 
a further precipitation of lead taurocholate. (Long). 

174. Add a few drops of oleic acid to 5 mils of bile in a test 
tube, shake well and at once place a drop of the mixture on a slide 
and examine, under the microscope, the numerous fatty globules. 
Place the test tube with the bile in a warm bath for an hour or so, 
shaking occasionally and then examine a drop with the microscope ; 
comparatively few fatty globules will be seen. The oleic acid has 
combined with the base of the bile-salts to form a soap. 

175. Prepare three test tubes as follows: (1) In one test tube 
put 5 mils of bile and a drop of oleic acid. (2) In another 5 mils of 
water. (3) In another 5 mils of bile. To each of the three add 
about 1 mil of fresh melted butter or lard. Shake well and place all 
three in a warm bath. Note in which tube the emulsion continues 
longest. 

176. Free fatty acids have the power of decomposing the bile 
salts with liberation of their acids. The emulsifying power of bile 
is slight ; but in the presence of fatty acids it forms soaps, which 
have a much greater emulsifying power. Animal membranes 
moistened with bile permit the passage of fatty oils, while if they 
are moistened with water only the oil cannot pass through. This 
is important in connection with certain digestive phenomena. 



XIII 
MILK 

177. Newly drawn milk is an opaque fluid of a white color. 
Its color and opacity are due to its being an emulsion, i. e., con- 
sisting of little globules of fat suspended in a solution of albumin, 
sugar and salts. When the milk is allowed to stand, the fat globules, 

56 



being lighter than the fluid in which they swim, rise in great part 
to the top and form cream, and part of the fluid often acquires a 
bluish tinge. It is said that a similar separation also takes place in 
the milk gland itself, so that the milk last drawn is richest in cream. 
The globules of fat are prevented from uniting by the thin albu- 
minous coating (the presence of this coating is denied by some) 
which surrounds each, but when this is broken by agitation, they 
coalesce, forming butter. Changes also occur in the milk sugar, 
casein and fats, more or less quickly, according to the higher or 
lower temperature to which the milk is exposed. The milk sugar 
becomes converted, apparently through the agency of a ferment, 
into lactic acid. This gives the milk an acid reaction, and precipi- 
tates the casein, causing the milk to curdle. The coagulum or curd, 
incloses the fat globules. The liquid from which it is separated, a 
solution of milk sugar and salts, is known as whey. The curd, when 
completely separated from the whey, is called cheese. 

The average composition of human and cow's milk and of the 
cream from cow 's milk is given in the following table : 

Human Milk Cow's Milk Cream 



Water 


88.20 


86.35 


73.35 


Proteins 


2.00 


4.40 


4.40 


Fat 


3.40 


4.40 


18.00 


Lactose 


6.00 


4.50 


4.50 


Inorganic Salts 


0.35 


0.75 


0.75 



178. Test the reaction of milk. Fresh cow's milk may often be 
neutral or even acid or amphoteric i. e., will color blue litmus red 
or red litmus blue. Sour milk is acid. The reaction of fresh human 
milk is alkaline. Some free lactic acid is present in the fresh milk 
of carnivora. 

179. Determine the specific gravity of unskimmed milk with an 
accurate lactometer. Allow some milk to stand until the next day. 
Remove the cream and again take the specific gravity, then add from 
10% to 25% of water and take the specific gravity once more. 

The laws of New York require milk to have a density of not less 
than 1.029, and total solids of not less than 11%%, of which 3% 
must be fats. 

Various forms of apparatus for testing milk are on the market, 

57 



A convenient apparatus is sold 'by the Whitall Tatum Co. of New 
York. (Apparatus No. 2). The following suggestions and direc- 
tions accompany each apparatus: The lactometer is intended to 
show the specific gravity or density of milk at the temperature of 
60° Fahrenheit. On the scale represents a specific gravity of 
1.000, which is that of water, and 100 represents a specific gravity 
of 1.029, which has been established as the lowest specific gravity 
of the milk from a healthy and ordinarily well-fed cow. 

The average lactometer reading of normal milk is 110, and all 
milk testing below 106 must be regarded as doubtful. 

Whenever milk shows less than 106, one of two things may be 
suspected : First, that the milk contains an unusual amount of 
cream; Second, that the milk has been watered and perhaps 
skimmed. 

It must not be assumed that milk of a low specific gravity is 
impure, for it may be rich in cream ; nor that milk of a high specific 
gravity is pure, for it may have been skimmed and its density in- 
creased by the removal of the fats. Color, taste and odor will 
indicate to some extent the quality, but, by the use of the cream- 
ometer the percentage of cream in a sample of milk can be deter- 
mined. 

The thermometer is required to determine the temperature of 
the milk since the lactometer is correct only when the milk is at 
60° Fahrenheit. The following rule for the correction of lacto- 
meter readings is sufficiently accurate for ordinary purpose^: 
For each 2% degrees of temperature above 60°, add one to the 
reading of the lactometer; example: Lactometer 114, thermom- 
eter 70°, or 10° above standard; add 4 to lactometer reading, 
making it 118. 

For each 2% degrees below 60°, subtract one from the reading 
of the lactometer; example: Lactometer 114, thermometer 55°, 
or 5° below standard; subtract 2 from the lactometer reading, 
making it 112. 

In testing milk, the following directions should be observed: 
Fill the creamometer nearly full with the milk; insert the lacto- 
meter and note carefully the point on the stem to which it sinks; 
take the temperature of the milk and correct the reading of the 
lactometer according to the rule given above. If the corrected 



lactometer reading is less than 106, or if it is suspected that the 
milk has been skimmed, fill the creamometer to the point marked 
and place it in a cool place, where it will not be disturbed for 
twelve hours ; in the summer it can be kept in the refrigerator. 
At the end of that time the cream will have risen and the per- 
centage can easily be read from the scale on the jar. The lowest 
safe proportion of cream is 15%, and a percentage lower than 
that will surely indicate that the milk was poor originally, or has 
been partly skimmed. 

180. Examine a drop of fresh cow's milk under the micro- 
scope. It consists of a clear fluid containing a large number of 
highly refractive fat globules. Let a drop of osmic , acid solution 
run under the cover glass; in a short time the globules become 
stained 'brown-black. 

181. To some milk in a test tube add a few drops of sodium 
or potassium hydroxide and heat. The liquid becomes yellow, 
then orange, and finally brown. 

182. To a 4% solution of lactose add some of the same reagent 
and heat. The same color is developed as in 181, which is due to 
the sugar present in the milk. 

183. To some unboiled or unpasteurized milk in a test tube add 
a few drops of fresh tincture of guaiac (20% solution in alcohol) ; 
agitate and add some peroxide of hydrogen (or old turpentine) . A 
blue color develops. The reaction is sometimes better if allowed to 
stand for a time. A similar result is given by the blood. 

184. Repeat the experiment with milk that has been boiled. 
The blue color is not given — due to changes in the protein. 

185. Mix 5 mils of fresh milk with 15 drops of neutral artificial 
gastric juice, and heat in the water bath to 40 o, C. In a short time 
the milk curdles so that the tube can be inverted without the curd 
falling out. By and by the whey is squeezed out of the clot. The 
curdling of milk by the rennin enzyme present in the gastric juice 
is quite different from that produced by the "souring of milk," or 
by the precipitation of casein by acidls. Here the casein (carrying 
with it most of the fats) is precipitated in a neutral fluid. 

186. To the same test tube after the above process add 10 mils 
of a 0.2% hydrochloric acid, and put into the incubator until the 
next exercise. Note any changes when next examined and test for 
peptones. 

59 



187. Dilute 5 mils of milk with 15 mils of water, add a little 
2% acetic acid and warm. A precipitate is formed. Filter and save 
both precipitate and filtrate. This precipitate is not the same as 
that obtained by rennet. The acid precipitate is casein and is freely 
soluble in dilute alkali, the rennet clot is paracasein and is much 
less soluble in dilute alkali. 'Cheese is made with rennin and cannot 
be made with acid. 

188. The filtrate obtained from 187 is to be divided into two 
portions. To the first portion apply Trommer's test. A red pre- 
cipitate indicates the presence of a reducing sugar — lactose. 

189. To the second portion of the filtrate apply the xantho- 
proteic reaction. An orange color represents the presence of a 
protein (lact-albumin). 

190. To the precipitate obtained from 187 add a little ether in 
a test tube and agitate for a few minutes. Pour off the ether upon 
some paper and note whether it leaves a permanent greasy stain 
indicating the presence of fat. 

191. To the residue left in 190 add a little dilute potassium 
hydroxide (0.1%). A solution is effected. Apply the xanthoproteic 
reaction to this fluid. An orange color denotes the presence of a 
protein — casein. 

192. The action of milk with pancreatic extract is somewhat 
complicated on account of the complexity of milk itself. The sugar, 
fats and proteins all undergo some change from the action of the 
different pancreatic enzymes. Perhaps the most interesting of these 
changes is that produced in the proteins, and is commonly called 
peptonization. The peptonization, or digestion, of milk is .quite 
often practised in the preparation of food for the sick room, and is 
illustrated by the following experiment. Dilute about 10 mils of 
milk with an equal volume of distilled water and add a half a gram 
of sodium bicarbonate. Then add a few drops of pancreatic extract, 
shake the mixture and keep at 40° C. on the water bath for about a 
half an hour. Then filter and apply the biuret test for peptones. 
The pancreatic extract from beef acts more strongly upon the pro- 
teins; that from the pig is very active in converting starch into 
sugar. 

193. Fill a test tube half full of milk and boil it. Add a tablet 
of rennin. Prepare another test tube, but use fresh unboiled milk 

60 



with a tablet of rennin. Place 'both tubes in the water bath at 38°C. 
After some minutes compare the tubes. The boiled milk should not 
be coagulated. The unboiled milk should be clotted. Leave this 
tube in the water bath if necessary, until the whey has separated 
quite completely from the curd. Filter and use the filtrate in the 
following tests. 

194. Test one portion of the whey filtrate by adding a few drops 
of nitric acid and a little ammonium molybdate solution and heat. 
A yellow precipitate indicates phosphates. Test another portion by 
adding a little silver nitrate. A white precipitate insoluble in nitric 
acid indicates chlorides (chiefly potassium and sodium) . To another 
portion add a little ammonium oxalate. A precipitate indicates 
calcium salts. Test the remaining portion for albumin using the 
xanthoproteic test. 

195. To a test tube half full of milk add 3 or 4 drops of a satu- 
rated solution of ammonium oxalate; mix, add a tablet of rennin 
and digest at 38°-40°C. for at least a half an hour. There should 
be no coagulum. Then add a few drops of a 2% solution of calcium 
chloride and digest again. Does the milk coagulate ? 

196. Separation of casein by salts. To a test tube half full of 
milk, add crystals of magnesium sulphate or sodium chloride to satu- 
ration. The casein and fat separate out, rise to the surface, and 
leave a clear salted whey beneath. Casein, like globulins, is precip- 
itated hy saturation with MgS0 4 or NaCl, but it is not coagulated 
by heat. It was at one time supposed to be an alkali albumin, but 
the latter is not coagulated by rennin. It appears to be a nucleo- 
albuniin — i. e., a compound of a protein with nuclein, the latter is 
a body rich in phosphorus. 

197. Boil a little milk in a small beaker or evaporating dish. 
There is no coagulation. A scum forms upon the surface which 
returns as often as it is removed. This is due chiefly to casein 
entangled in protein drying on exposure to air. 

198. Place a small quantity of milk in a warm place for one or 
two days ; then test the reaction, it will be found to be acid ; this is 
due to fermentation, in the process of which the milk sugar is con- 
verted into lactic acid. 



61 




Fig. 1. The left side of the 
figure represents the appearance 
of" milk with the ordinary micro- 
scope. The dark field at the 
right shows the ffalactomicrons. 



199. Milk under the Darkfield Microscope. The special appa- 
ratus involved in connection with the darkfield microscope has made 
it possible to see in milk very minute paricles which are not visible 

with the ordinary microscope. These 
minute particles show active Brownian 
movement. With undiluted milk there 
is a sufficient amount of material pres- 
ent in the microscopic field to reflect 
so much light that a bluish-gray or 
slate colored background results. This 
difficulty may be overcome by diluting 
the milk with water in the proportion 
of one to one hundred. The compo- 
sition of the particles has not yet been 
determined with precision. It has 
been suggested that they are composed 
of protein material, possibly casein in 
suspension since they disappear or 
are diminished in number after milk has been digested with certain 
proteolytic enzymes. Our own experiments show that after prote- 
olytic digestion has taken place many of the particles remain and 
the possibility of the existence of two kinds of particles is suggested ; 
one group representing protein — probably casein, and the other 
perhaps representing fat particles. During fat absorption in the 
digestive tract the chyle and blood show a very great number of 
minute particles (chylomicrons) . In these fluids there is the strong- 
est evidence to show that these particles are composed of fat. The 
chylomicrons soon become much reduced in number in the blood and 
it is not impossible that their disappearance, in part, is associated 
with their passing over into the secretion of the mammary gland. 

The fat globules of the milk have a characteristic appearance 
with the darkfield microscope; some of them approach the particles 
in minuteness of size but show a difference in their power of refract- 
ing the light if carefully examined. For the minute particles 
present in milk the term galactomicrons is suggested. 



62 



XIV 
BLOOD 

200. The blood is a red, thick, opaque fluid. The specific gravity 
varies from 1.045 to 1.075 with an average for adult beings of about 
1.055 ; it depends primarily upon the amount of hemoglobin present. 

For examination, it is convenient to consider the blood as com- 
posed of two parts : The corpuscles and the protein liquid in which 
they are suspended — the plasma. The solid blood corpuscles in man 
may constitute nearly one-half the weight of the blood. In some 
animals as the ox, they make up but one-third of the weight of the 
blood. 

The color of the blood is caused by the red corpuscles. Even 
comparatively thin layers of the blood are opaque from their pres- 
ence. The coloring matter (hemoglobin) can be set free from the 
corpuscles by water or by many chemical reagents.' The color then 
becomes much darker, since the light is no longer reflected from the 
surface of the corpuscles. The addition of strong neutral salt 
solutions to blood turns it bright red, because of the increased reflec- 
tion of light from the shriveled corpuscles. 

201. Fresh blood may be obtained and denbrinated at a 
slaughter house, and a few drops of formalin added to it will prevent 
putrefaction for some time. It is better, however, when possible, 
to obtain the blood by bleeding an animal. After the dog, or any 
other animal of convenient size, has been anesthetized, the carotid 
or femoral artery is exposed and isolated from surrounding parts 
for an inch or two of its length, and a clamp or ligature applied to 
the proximal portion of the artery, i. e., as far as possible toward the 
heart. Apply another clamp about one inch distally and between 
the clamps make an incision in the artery and insert a glass canula 
and tie it tightly in place. Remove the proximal clamp and the 
blood will pass through the canula, and the animal allowed to bleed 
to death. When the animal is apparently dead, an interesting 
experiment may be performed by injecting into the artery some 
normal salt solution of the same temperature as the body and note 
the reviving effect. 

202. The blood obtained as above directed is to be caught in 
four different vessels and each portion is to be treated as follows: 

63 



One portion of the blood is to b,e defibrinated by immediate whipping 
with some broom straws tied in a small bundle and the fibrin as it 
collects on the straws is to be saved for future use. The defibrinated 
blood is also to be reserved for later study. Another portion of the 
blood is to be collected in a flask and the phenomenon of clotting or 
coagulation observed. Another portion of the fresh blood is to be 
mixed with an equal volume of saturated solution of sodium sul- 
phate. And still another portion into a solution of potassium 
oxalate in the proportion of 1 of the oxalate to 4 of the blood. 

203. Test the reaction of b,lood by pricking one of the fingers 
behind the nail. Put a drop of the blood on a piece of ordinary 
litmus paper which has been soaked in salt solution. The substances 
on which the alkaline reaction depend will diffuse out in a ring 
around the drop, while the hemoglobin remains in its original 
position. Modern methods in physical chemistry indicate that the 
blood has a neutral reaction. The reaction of a solution is deter- 
mined by the proportion of hydroxogen and hydroxyl ions. If the 
hydrogen ions are in excess, the fluid is acid. If the hydroxyl 
ions predominate, the reaction is alkaline. 

204. Place a thin layer of defibrinated blood on a glass slide ; 
try to read printed matter through it. The blood is too opaque and 
the print cannot be read, the light is reflected from the corpuscles 
in all directions and but little passes through. 

205. Place 1 mil of defibrinated blood in a test tube and add 
5 mils of distilled water, and warm slightly. Note the change of 
cdlor by reflected and transmitted light. By reflected light it is 
much darker — almost black, but by transmitted light it is trans- 
parent. This constitutes "laky" blood due to the withdrawal of 
the hemoglobin from the red corpuscles into the water. Test the 
transparency by looking at some printed matter through this blood 
as in 204. 

206. To 2 mils of defibrinated blood in a test tube add 5 volumes 
of a 10% solution of sodium chloride. It 'Changes to a very bright, 
florid, brick-red color. Compare its color with No. 205. 

207. Place a watery solution of defibrinated blood in a dialyzer 
or parchment tube, and suspend in a vessel of distilled water. After 
several hours note that no hemoglobin has passed into the water. 
Test the diffusate for chlorides with silver nitrate and nitric acid. 
Hemoglobin does not dialyze although it is crystaJllizable. 

64 



208. Put a drop of blood on a slide with a small crystal of 
common salt and a drop or two of glacial acetic acid. Put on a 
cover-glass. Heat gently over a flame. Take the slide away from 
the flame for a few seconds, then heat it again for a moment, and 
repeat this process for a few times. Now let the slide cool and 
examine with the microscope (high power). The small black or 
brownish-black crystals of hemin will be seen. This test is often 
important in some medico-legal cases where only a trace of blood is 
available for examination. If the blood stain be upon a piece of 
cloth, it may be soaked in a little distilled water and examined by 
the spectroscope or micro-spectroscope. The liquid may then be 
evaporated to dryness on the water bath and the hemin test made. 
Or perform the hemin test directly on the piece of cloth. 

209. In the blood saved for clotting, note that in a few minutes 
the blood congeals, and when the vessel is tilted the blood no longer 
moves as a fluid, but as a solid. After an hour or so, pale yellow 
colored drops of fluid — the serum — are seen on the surface, having 
been squeezed out of the red mass, the latter being the clot and 
consisting of fibrin. 

Note in the clot of horse's blood the upper li^ht colored layer 
of leucocytes — the buffy coat. Coagulation is slow in this animal 
and the red and white corpuscles on account of the difference in 
their specific gravity have time to separate. 

210.' Salted Plasma. Note that in the flask containing the 
mixture of blood and sodium sulphate, no coagulation has occurred. 
Place some of this fluid in the centrifuge to separate the eorpuesles 
and plasma, or let the mixture stand until the corpuscles sink ; the 
plasma mixed with the saline solution is known as the salted plasma. 

211. Oxalate Plasma. Note also that the potassium oxalate 
blood mixture does not coagulate. Centrifuge the mixture or let 
stand until the corpuscles fall, to obtain the plasma. The oxalate 
combines with the calcium which is necessary for coagulation. 

212. To a portion of the oxalate plasma add a few drops of a 
2% calcium chloride solution. Coagulation results (more quickly 
at 40°). 

213. To another portion of the plasma add a little fibrin-ferment 
prepared by the demonstrator. The fibrin-ferment is prepared as 
follows: Take fresh fibrin, wash it under a tap with water (best 

65 



in a piece of cotton) until perfectly colorless. Squeeze out the water 
and cover the fibrin with an 8% solution of sodium chloride. After 
a few hours, if the solution is filtered it will show the presence of 
the ferment. 

Another method is: Precipitate some blood serum with about ten times its 
volume of alcohol. Let it stand for several weeks, then extract the precipitate 
with water. The water dissolves out the fibrin-ferment, but not the other coag- 
ulated proteins. 

214. Add a drop of freshly prepared tincture of guaiaeum to a 
small amount of diluted defibrinated blood, and then some hydrogen 
peroxide or old oil of turpentine. The color changes to blue. This 
is often used as a test for hemoglobin, but other substances (oxygen 
carriers) give a blue color under the same conditions. 

215. Place some hydrogen peroxide over fresh fibrin in a watch 
glass; bubbles of oxygen are given off. 

216. Immerse a flake of fibrin in freshly prepared tincture of 
guaiacum, (5% of pure resin in alcohol) and then immerse the flake 
in hydrogen peroxide. A blue color is developed, due to the ozone 
liberated by the fibrin and forming a blue color with the resin. 
Compare 214. 



XV 
BLOOD 

217. Protein reactions. Dilute 5 mils of serum with 35 mils of 
water. Add a little litmus solution to color and neutralize with 
0.2% hydrochloric acid. Is alkali albumin present? 

218. To a separate portion of serum add a little acetic acid 
and heat. 

219. Apply the xanthoproteic reaction to another portion of 
serum. 

220. Acidify another portion strongly with acetic acid and add 
a few drops of a solution of f errocyanide of potassium. 

221. To separate portions apply Millon's reagent and Piow- 
trowski's test (6). 

66 



222. To another portion add a little alcohol. 

223. Saturate another portion with ammonium sulphate. This 
precipitates all of the proteins, globulin and albumin. Filter. The 
filtrate does not respond to any of the tests for proteins, 

224. To another portion of the diluted serum add a little silver 
nitrate solution. A white, curdy precipitate forms, soluble in 
ammonia but not in nitric acid. Chlorides are present. 

225. In the following tests use separate portions of the serum 
for each test. Add barium chloride. A white, heavy precipitate 
insoluble in nitric acid. Sulphates are present. 

226. Add nitric acid and molybdate of ammonia and heat. A 
yellow precipitate indicates the presence of phosphates. 

227. Test with Fehling's solution, and boil. Red, cuprous 
oxide indicates a reducing sugar — dextrose. 

228. To a little of the defibrinated blood in a test tube add a 
few drops of sulphuric acid. Stir up the solution and note the 
peculiar odor of blood, intensified by the liberation of traces of 
volatile acids by the sulphuric acid. 

229. Detection of paragl'obuTin (fibrinoplastin, or serumglobulin) . Pass 
some 00 through a beaker of dilute serum for 20 minutes or more.' (The CO o 
may be generated by the action of dilute hydrochloric acid upon small pieces 
of marble in a jar and the gas conveyed to the beaker). Let the precipitate 
settle. It is paraglobulin. Decant and, after washing with water, dissolve 
some of it in a little dilute saline solution, use Piowtrowski 's test and prove it 
a protein. 

230. Take equal quantities of blood and ether in a test tube. 
Shake thoroughly and let the ether separate. Then pour the ether 
into a watch-glass or evaporating dish and when evaporated examine 
for globules of fat. 

231. Evaporate a little b,lood to dryness in a crucible or evap- 
orating dish. Raise the temperature to red heat to convert the blood 
to ash. When cool add a little nitric acid, heat, dilute with water 
and filter. Make the following tests with the filtrate : 

232. To a small portion of the filtrate add a little sulphocyanide 
of potassium. A red color indicates iron. 

233. To another portion add a little ammonium molybdate 
solution. A yellow precipitate, after allowing the mixture to stand 
for some time, indicates phosphates. 

67 



234. To another portion add a little silver nitrate solution and 
a drop or two of nitric acid. A white, cloudy precipitate indicates 
chlorides. 

235. Examination of blood with a spectros'cope. With a small 
direct vision spectroscope focus on the sky or bright light until the 
spectrum shows clearly. Narrow the slit until the spectrum is as 
distinct as it can be made. Hold the spectroscope so that the red is 
at the left of the field. Dip a wire into some water, and then into 
some salt or sodium carbonate, and hold it in a flame of a fish-tail 
■burner. Note the change in the spectrum. 

236. Arrange the apparatus with the aid of a demonstrator, so 
that the spectroscope, gas-flame and substance to be examined, are 
in their proper relations. Half fill the vial or test tube with 
defibrinated blood. Nothing can be seen until the blood is properly 
diluted. Continue diluting until two bands of oxyhemoglobin 
appear in the spectrum. Note their position, and which one dis- 
appears first when the solution is diluted far enough. 

237. Add a drop or two of ammonium sulphide solution or 
Stokes' fluid to reduce the oxyhemoglobin. Note the result. 

238. Pass some illuminating gas through some blood for a con- 
siderable time. Examine with a spectroscope. Add a drop or two 
of ammonium sulphide or Stokes' fluid. Compare this with 235. 




Fig. 2. The diagram at the left shows the appearance of the blood after 
fasting when only a few chylomicrons are present. The diagram at the right 
shows chylomicrons in great numbers after the digestion and absorption of fat. 
(Gage). 

68 



239. Appearance of blood with the darkfield microscope. The 
red and white corpuscles are clearly visible as objects reflecting the 
light strongly but without color. If the blood be observed after a 
meal, containing fat, has been digested, there will be seen a very 
great number of minute particles or chylomicrons (Gage). These 
were formerly called hemoconia or blood dust. Recent evidence 
has shown that they are associated with fat absorption and have no 
relation to broken down blood elements or debris. A strictly protein 
diet or a strictly carbohydrate diet contributes nothing to the 
presence of the chylomicrons in the blood, but a fat diet causes a 
very marked increase in their number. After fat absorption has 
occurred the greatly increased number of chylomicrons becomes 
markedly reduced in the course of a few hours. 



69 



PART II 



Experimental Physiology 



XVI 

DISSECTION OF FROG'S HEART, VAGUS AND SCIATIC 
NERVES AND LEG MUSCLES 

Each dissection is to be carefully demonstrated to one of the 
instructors before beginning the dissection of a new part. 

240. Dissection of Frog's Heart. With a pair of strong 
scissors and forceps cut through the pectoral girdle. Remove the 
sternum and expose the heart, cut carefully through the pericardium 
and note the division of the heart into ventricle, auricles, and 
truncus arteriosus. (Do not remove the heart until the vagus nerve 
has been dissected). 

Ca v'»Ty <>i /^^^^^^^a^^v 






Valve y 



)... Valve. 

...CaYi Ty *\ 
ve nT-ricle, 



Fig. 3. Dissection of the frog 's heart showing the relationship of the cav- 
ities and the principal blood vessels. 

Raise the apex of the ventricle slightly and note a delicate band 
of connective tissue binding the ventricle to the body, (the frenum) . 
The ventricle is of a conical shape and is usually of a paler color 
than the auricles. It has thick walls. 

The auricles are two in number although externally the division 
is not easily apparent. The right is larger than the left and both 
are usually engorged with blood. The walls of the auricles are thin. 

71 



The truncus arteriosus is a cylindrical tube somewhat swollen as 
it lies upon the auricles. The truncus soon divides into two arches, 
one passing to the right, the other to the left. Each arch soon splits 
into three vessels, the carotid, for the head, the pulmocutaneous, 
carrying the venous blood to the lungs and skin to be oxygenated, 
and the aorta, which curves around to the back to meet its fellow with 
which it unites to form the descending aorta. Lift up the ventricle 
and make out the following structures: The right and the left 
superior vena cava, bringing back blood from the head and 'upper 
extremities ; the inferior vena cava, appearing just above the liver ; 
the sinus venosus, (practically a fusion of the venae cavae), the 
chamber into which the cava! veins open. The sinus in turn com- 
municates with the right auricle. 



Hypoglossal ... 
>i?rve 



vajuj ntrYQ, 



Oesof>hajeal._.. 






Sinus— 
venosus 

vein." 




*T>un.eus va«u$ 



Fig. 4. Lateral aspect of the 'heart, showing its principal parts and the 
distribution of the branches of the vagus nerve. 



Carefully slit the heart lengthwise into ventral and dorsal halves. 
In the ventricle note the comparatively small size of the cavity and 
the thick walls ; note the two openings in the ventricular cavity — 
one from the auricles, guarded by auriculo-ventricular valves, the 
other continuous with the truncus arteriosus ; note in the truncus at 
its base near the ventricle three small semilunar valves, also a longi- 
tudinal fold or so-called spiral valve. The swollen portion of the 
truncus is known as the pylangium; the distal portion formed by 
the fusion of the aortic arches is known as the synangium. 

241. Dissection op the Vagus Nerve. Introduce a glass rod 
into the frog's throat to distend the parts. Beginning at the angle 
of the mouth, remove the skin between it and the arm and tym- 

72 



panum. The projection formed by the articulation of the lower and 
upper jaws may be cut off. Remove the arm. Dissect away the 
muscles and expose the scapula ; this in turn may be tilted to one 
side or removed. In front of, and partially under the scapula 
nerves may be seen. Two nerves will be found lying close together 
and accompanied by a blood Vessel. The first of these nerves is the 
glossopharyngeal, the second the vagus with its branches. Follow 
both nerves toward the cranium and note that both glossopharyngeal 
and vagus emerge from the cranium through the same foramen. 

Dissect the glossopharyngeal distally and note that it supplies 
the tongue. 

Dissect the vagus noting that it gives off a branch to the oeso- 
phagus, then a smaller one the laryngeal curving around the aorta 
to supply the larynx, and finally branches to the lungs and heart. 
In the turtle the parts are larger and more satisfactorily demon- 
strated. 



fie cVm s - ^%v ^H^ W 

G Tflcilisj'"" ^^r j 

§/■ /jJt * E;rTe n s ° r 

Tibials 'J^/fj/y Cr ^x\% 



"PosTicus 



"Ti'bialis 



Fig. 5. Ventral aspect of the superficial muscles of the left leg of the frog. 

242. Dissection of Muscles of Frog's Thigh and Leg. Ventral As- 
pect. Remove the skin from the ventral aspect of the leg and expose the super- 
ficial muscles. 

Sartorius, a long narrow muscle crossing the thigh obliquely from the 
outer to the inner side. It arises from the iliac symphysis below the acetabulum 
and is inserted into the inner side of the head of the tibia, 

73 



The Adductor Magnus is a large muscle lying along the inner border of 
the sartorius but passing beneath it at its distal end.. Its origin is from the 
pubic and ischial symphyses, and the muscle passes under the sartorius to be 
inserted into the distal third of the femur. 

The Adductor Longus is a long, thin, narrow muscle lying along the outer 
side of the adductor magnus and often completely hidden by the sartorius; its 
origin is from the iliac symphysis beneath the sartorius and unites a little way 
beyond the middle of the thigh with the adductor magnus. 

The Rectus Internus Major or Gracilis, is a large muscle lying along the 
inner side of the adductor magnus and of the sartorius. Its origin is from the 
ischial symphysis and it is inserted into the head of the tibia. 

|Ji . GtuTe u-s. 



Vastus 



^'J& Cloaca, 

^5^* *Se m \ m € m fer* n#$ u$ ( 
— Biceps. 
Rectus inlernws 



-Gas trocnemi u£, 

«... TfiToneus 
jY--Tt-biali.s anlicuj 



Fig. 6. Dorsal aspect of the superficial muscles of the left leg of the frog. 

The Rectus Internus Minor is a narrow, ribbon-like muscle passing along 
the inner or flexor margin of the thigh ; it arises from a tendinous expansion 
connected with the ischial symphysis and is inserted into the inner side and just 
below the head of the tibia. 

243. Dorsal Aspect of the Thigh. The Triceps Extensor Femoris is 
the great extensor muscle of the thigh; it arises by three distinct origins from 
the ilium and acetabulum and is inserted into the tibia just below its head. 

The Rectus Anticus Femoris is the middle division of the triceps; it arises 
from the ventral border of the posterior third of the ilium in front of the acet- 
abulum; about half way down the thigh it joins the next division. 

The Vastus Internus is the ventral head of the triceps and lies between the 
sartorius and the rectus anticus. It arises from the ventral and anterior border 
of the acetabulum. 

74 



The Vastus Ext emus is the dorsal head of the triceps. It arises from the 
posterior edge of the dorsal crest of the ilium and joins the other two divisions 
of the triceps at about the junction of the middle and distal thirds of the thigh. 

The Gluteus lies in the thigh between the rectus anti'cus and the vastus 
externus. It arises from the sacrum and is attached to the femur. 

The Biceps is a long, slender muscle arising from the crest of the ilium 
just above the acetabulum. It lies in the thigh along the inner border of the 
vastus externus and is inserted by a flattened tendinous expansion into the distal 
end of the femur and the head of the tibia. 

The Semimembranosus is a stout muscle lying along the inner side of the 
biceps, between it and the rectus intemus minor. It arises from the dorsal 
angle of the ischial symphysis just beneath the opening of the cloaca and is 
inserted into the back of the head of the tibia. There is an oblique line of 
tendinous intersection running obliquely through its middle. 

The Pyriformis is a slender muscle which arises from the tip of the urostyle, 
passes backwards and outwards between the biceps and the semimembranosus 
and is inserted into the femur at the junction of its proximal and middle thirds. 

244. Ventral Aspects of the Deep Muscles of the Thigh. The Scmi- 
tendinosus is a long, thin muscle which arises by two heads-, an anterior one from 
the ischium close to the ventral angle of the ischial symphysis and the acetab- 
ulum ; and a posterior one from the ischial symphysis. The anterior head passes 
through a slit in the adductor magnus and unites with the posterior head in the 
distal third of the thigh. The tendon of insertion is long and thin and joins 
that of the rectus intemus minor to be inserted into the tibia just below its 
head. 

The Adductor Brevis is a short wide muscle lying beneath the upper end 
of the adductor magnus. It arises from the pubic and ischial symphyses and 
is inserted into the proximal half of the femur. 

The Pectineus is a smaller muscle lying along the outer (or extensor) side 
of the adductor brevis. It arises from the anterior half of the pubic symphysis 
in front of the adductor brevis and is inserted like it into the proximal half of 
the femur. 

245. Dorsal Aspect of the Deep Muscles of the Thigh. The Ilio- 
psoas arises by a wide origin from the inner surface of the acetabular portion 
of the ilium; it turns around the anterior border of the ilium and crosses in 
front of the hip joint, where for a short part of its course it is superficial be- 
tween the heads of the vastus intemus and of the rectus anticus femoris; it 
then passes down the thigh beneath these muscles and is inserted into the back 
of the proximal half of the femur. 

The Quadratus Femoris is a small muscle on the back of the upper part of 
the thigh; it arises from the ilium above the acetabulum and from the base of 
the iliac crest ; it lies beneath the pyriformis and is inserted into the inner 
surface of the proximal third of the femur, between the pyriformis and the 
iliopsoas. 

The Obturator is a deeply located muscle which arises from the whole length 

75 



of the iliac symphysis and the adjacent parts of the iliac and pubic symphyses 
and is inserted into the head of the femur close to the gluteus. 

246. Muscles of the Tibial Portion of the Leg. The Gas- 
trocnemius is the large muscle forming the calf of the leg; it has two 
heads of origin, the larger of which arises by a strong flattened 
tendon from the flexor surface of the distal end of the femur ; while 
the smaller head which joins the main muscle about one-fourth of 
its length below the knee, arises from the edge of the triceps femoris 
where it covers the knee. The muscle is thickest in its upper third 
and tapering posteriorly ends in the strong Tendon of Achilles, 
which passes under the ankle joint, being much thickened as it does 
so and ends in the strong planter fascia of the foot. 

The Tibialis Posticus arises from the whole length of the flexor surface of 
the tibia; it ends in a tendon which passes around the inner malleolus, lying- in 
a groove in the lower end of the tibia and is inserted into the dorsal surface of 
the astragalus. 

The Tibialis Anticus lies on the extensor surface of the leg; it arises by 
a long thin tendon from the lower end of the femur and divides about the middle 
of the leg into two bellies which are inserted into the proximal ends of the 
astragalus and calcaneum respectively. 

The Extensor Cruris lies along and is partly covered by the tibialis anticus. 
It arises by a long tendon from the condyle of the femur and runs in a groove 
in the upper end of the tibia and is inserted into the extensor surface of the 
tibia along nearly its whole length. 

The Teroneus is a stout muscle which lies between the tibialis anticus and 
the gastrocnemius. It arises from the distal end of the femur and is inserted 
into the outer malleolus of the tibia and the proximal end of the calcaneum. 

247. Dissection of the 



ferntxr 




Wzrue m usclc p reparation. 
Fig. 7 



Sciatic Nerve. Expose the 
muscles of the dorsal aspect 
of the thigh, carefully sepa- 
rate the biceps and semimem- 
branosus; closely applied to 
the deeper margin of the 
biceps will be found the 
sciatic nerve accompanied by 
a blood vessel. Carefully 
follow the nerve toward the 
b,ody noting its passage be- 
tween the pyriformis and the 
head of the biceps. Follow 



76 



the nerve up to its connection with the lumbar region of the spinal 
cord. 

Return to the middle of the thigh and note that the nerve sends 
oft' a branch which passes along the extensor side of the tibia along 
the peroneus and beyond. Follow the sciatic and note that at the 
knee joint it again divides, one branch going to the gastrocnemius 
and the other to the tibialis posticus muscle. The sciatic nerve, 
gastrocnemius muscle and a portion of the femur comprise a nerve- 
muscle preparation. 



XVII 
HEART BEAT OF FROG 

248. Place a frog on its belly and note the movements of the 
caudal lymph-hearts. They are situated between the hip-joint and 
the median line in a slight depression. The contractions of these 
hearts are usnally visible through the skin, but are seen more dis- 
tinctly if the skin is removed without injury to the heart. 

Later, note that the lymph-hearts cease to beat after the destruc- 
tion of the caudal portion of the myel (spinal cord). 

249. Pith the frog. This is accomplished by severing the brain 
from the myel with a thin-hladed knife at the point where the 
cranium articulates with the atlas. A slight depression will be felt 
at this point, which will serve as a guide for the operation. The 
frog may be firmly held if wrapped in one corner of a towel. 

250. After pithing, lay the frog on its back and cut through the 
skin on the mid-line, and from the middle of this cut make lateral 
incisions through the skin. Raise up the end of the sternum and 
cut, a little to one side of the mid-line, through such parts as may 
be necessary to expose the heart. Pin the parts on the side and note 
the heart beating with some force and regularity. Count the number 
of heart beats per minute. Pinch up the pericardium with a pair 
of fine forceps and remove it from the heart. Tilt up the apex of 
the ventricle and note a small band of connective tissue passing from 
its dorsal surface to the adjoining wall of the pericardium. Seize 
this band with the forceps and divide it between the forceps and the 
pericardial wall. Connect the apex of the ventricle with a heart 

77 



lever and take a tracing of the heart beat npon the kymograph 
(revolving drum). Use also the apparatus for taking a time trac- 
ing. After immersion in the varnish and drying, paste the tracing 
in your notes. Lift up the apex of the ventricle, by means of the 
band already described, and with a sharp pair of scissors cut through 
the right and left aortae, the pre and post caval veins, and the sur- 
rounding tissue, taking care not to injure the sinus venosus. Place 
the heart in a watch-glass, moistening occasionally with physiological 
saline solution. The beats will not be interrupted at all, or for a 
very short time only. 

251. Watch the beating of the heart. Do the auricles and 
ventricle contract simultaneously ? What are the number of beats 
per minute ? Compare with 250. Then place in cold saline solution, 
count 'again. Then gradually heat, but not too high, and note the 
effect upon the rate of the heart beat. 

252. Lift up the apex of the ventricle, and with the scissors cut 
off the apex at the upper third of the ventricle. Watch the separated 
portions. Is there any difference in the beating? 

253. With the scissors separate the two auricles from each other, 
letting the attached portion of the ventricle remain to each auricle. 
Do they continue to beat ? 

254. The same frog, if it has been kept in a moist place, may 
be used for the following cilia experiment: Place the frog upon 
its back, and cut through the lower jaw, along the midline, con- 
tinuing the incision down the eosophagus as far as the stomach. 
Pin the parts back and moisten the mucosa with physiological salt 
solution, if it is at all dry. Place a small, thin piece of cork upon 
the mucosa just below the orbits, and note that the cork is carried 
toward the stomach by the cilia. Warm a little of the physiological 
salt solution to 30° C, and repeat the experiment. Apply heavier 
bits of substance to the mucosa, and note if their positions are 
changed. Apply to the strip of mucosa a few drops of a saturated 
aqueous solution of chloretone and note whether the motion of the 
cilia is affected or not. With a scalpel scrape some of the mucosa 
and examine the ciliated cells in the saline solution under the micro- 
scope. 

255. If the caudal lymph-hearts are still beating, pass a tracer 
or piece of wire down the spinal canal to destroy the myel. If 
thoroughly destroyed the lymph-hearts will cease to beat. 

78 



xvin 

THE CIRCULATION OF THE BLOOD 

256. This may 'be shown very nicely in the delicate external gill 
filaments of the Necturus, or in the tail of a tadpole, or in the web 
of a frog's foot which does not contain too much pigment. The 
animals should be injected with a few drops of a 1% solution of 
curare, in order that they may not move, and arranged upon the 
stage of the microscope, so that the parts to be examined may come 
clearly into the field of vision. Precautions should be taken against 
drying, by keeping the animal. well surrounded with moist cloth or 
absorbent cotton. 

257. If the frog is more convenient, prepare it by destroying 
the brain and injecting the curare under the skin of the back. Place 
the frog on its belly on the frog board and pin out the digits so that 
the web will be slightly on the stretch. Keep the parts moist. Put 
a very small drop of water upon the web, and cover it with a tri- 
angular piece of cover-glass, being careful that it does not cut the 
digits and that no fluid flows over its surface. Examine first with 
a low power, and then, if possible, with a high power. 

258. Note the course of the blood from the arteries to the veins. 
Arteries may be distinguished from veins by the fact that the blood 
corpuscles scatter to enter the capillaries diverging from the artery, 
while in the veins the corpuscles accumulate from the capillaries 
converging to form the vein. A slight pulsation may sometimes be 
observed in the smaller arteries. 

259. Note the greater velocity of blood in the arteries than in 
the veins; the individual corpuscles cannot, perhaps, be made out 
in either. 

260. Note the axial and peripheral zones in the arteries and 
veins ; the peripheral zone is small and under a low power appears 
free from corpuscles ; under a high power a few leucocytes may be 
seen in the peripheral zone, if the current is not too rapid ; in that 
of the veins a few leucocytes and occasionally a red cell will be seen 
moving along comparatively slowly. 

261. Note the passage of the corpuscles usually in single file 
through the capillaries. 

79 





Fig. 8 



Fig. 9 





Fig. 10 



Fig. 11 



Figs. 8 to 11. Fig. 8 — Leucocytes sending forth processes which penetrate 
the wall of the vessel.. Fig. 9. — Leucocytes partly through vessel wall, showing 
constriction in centre. Fig. 10 — Leucocytes after penetrating wall regain 
former shape. Fig. 11. — Appearance of vessel and surrounding tissue after 
diapedesis has gone on for some time. (After Craig). 



Blood. Capillary. 



a c d e / a 

Fig. 12. Diagrammatic Representation of the Manner in which a Leucocyte 
Traverses the Wall of a Capillary Blood-Vessel. (After Craig), a, Leucocyte 
before penetrating; o, leucocyte sending off process and granules beginning to 
withdraw to farther end of cell; c, leucocyte partly through wall, granules at 
upper end of cell; d, granules passing through the wall; e, granules arranged 
in the portion of the leucocyte farthest from vessel; /, leucocyte, after pene- 
tration, resuming its original condition; g, leucocyte swept from wall, showing 
retention of the clear penetrating process. 

80 



262. Note the elasticity of the red corpuscles, observing the way 
in which they bend and later regain their normal form. 

263. Study of inflammatory conditions. Remove the cover- 
glass and absorb the fluid on the web ; touch the middle of the web 
with the tip of a glass rod that has been dipped in creosote (or a 
2% solution of croton oil in olive oil) leaving a minute drop on the 
web. Put on a cover-glass as before and examine with the micro- 
scope. If not successful with the web, try the tongue or mesentery. 

264. Note the dilation of the arteries, the more distinct 
appearance of the capillaries, and the enlargement of the veins, 
accompanied by a quickening of the current. 

265. Note a little later, the slowing of the current, the vessels 
remaining dilated. 

266. Note that the leucocytes increase in number in the per- 
ipheral zone of both arteries and veins ; in the latter the leucocytes 
begin to cling to the sides, temporarily at first and then permanently. 
In the capillaries the leucocytes and, less frequently, the red cor- 
puscles stick to the capillary walls, partially or completely blocking 
the way. Later stagnation may set in and there is then the appear- 
ance of the gradual obliteration of the outlines of the corpuscles. 

267. Note the migration of the leucocytes from the capillaries 
and veins. This occurs when the circulation becomes slow. AVatch, 
at intervals of 10 minutes, some particular leucocyte adhering to 
the wall of a capillary or vein. 

268. Note the diapedesis of the corpuscles from the capillaries, 
seen to the best advantage in those capillaries in which the current 
has almost ceased. 

269. Note that the above effects are local, are of greatest intens- 
ity in the spot touched, that they extend some distance around the 
spot, but the circulation in the rest of the web is normal. If the 
injury has not been too severe, the circulation may become re- 
established in the stagnated spots, and the inflammatory appearances 
disappear. 

270. Pin out the two horns of the tongue and observe under 
the microscope. The tongue is at first pale but soon becomes red- 
dened as the vessels become filled with blood. With a low power 
the peripheral zone in the arteries and veins may probably be seen 
better here than in the web. 

81 



271. Place the frog on its back, cut through the skin and muscles 
on one side and draw out the mesentery and pin out a loop of it 
under the field of the objective and observe the circulation. The 
inflammatory phenomena can be well seen in this preparation or 
that of the tongue. (270). 



XIX 
REFLEX ACTION. GALVANI'S EXPERIMENT 

272. Experiments in reflex action. Pith a frog and place it 
on its belly. Note the position of its fore and hind limbs. Note the 
position of the head as compared with a normal frog. Are there any 
respiratory movements at the nostrils or throat ? 

273. Pull, very gently, one of the hind limbs into an extended 
position and then let go. Does it return to its former location ? 

274. Gently tickle one flank with a feather or blunt needle. Is 
there any contraction of the muscles ? 

275. Pinch the same spot sharply with a pair of forceps. Is 
there any movement of the leg of the same or opposite side ? 

276. Pinch the skin around the anus with a pair of forceps. 
What is the effect upon the legs ? 

277. Place the frog on its back. Does it make an effort to get 
into a natural position ? Does it show any sense of equilibrium ? 

278. Pass a hook through its lower jaw and hang it to the ring 
of a retort stand. How do the hind-limbs behave ? 

279. Pinch very gently the tip of one of the toes; what is the 
effect ? 

280. Fill two glasses, one with dilute sulphuric acid, the other 
with water. liaise the glass containing the acid, until the acid just 
touches the tip of the toes. Is the foot withdrawn? If so, raise 
the second glass and let the foot be immersed in it, to wash off the 
acid. 

281. Cut a small piece of filter or blotting-paper, moisten it 
with strong acetic acid and place it on the flank of the animal. 
What is the effect upon the leg? Put the piece of paper upon the 
opposite flank and hold the leg so as to prevent it from moving? 
Is there any action of the opposite leg? 

82 



282. Place similar pieces of paper upon different portions of 
the body. Note any variety of movements and what seems to be 
their purpose. 

283. Remove the frog from the hook and plunge it in a basin 
of water. This will wash off the acid. Does the frog make any 
movements in the water? Does it float? 

284. Inject 3 to 5 minims of 0.2% strychnine solution under 
the skin of the frog's back. Let it remain for a few minutes and 
then note the effect of the slightest stimulus, such as jarring the 
table upon which it lies. Then give 10 to 20 minims of 10% chloral 
hydrate with a pipette. Make sure that the fluid reaches the 
stomach. Note if there is any effect npon the convulsions, 

285. With a tracer or piece of wire destroy the my el, the con- 
vulsions cease. Try any of the preceding stimuli upon the frog now 
and note the result. 

286. Make a nerve-muscle preparation of one of the hind limbs. 
Dissect away the skin and muscles upon the dorsal aspect of the leg, 
until the sciatic nerve is exposed, leaving it connected with the 
lumbar plexus. Denude the femur of its muscles, using the greatest 
care not to injure the sciatic nerve. Keep the nerve moist with 
normal salt solution. Pass a copper hook under the sciatic nerve 
and hang to a tripod. Tilt the tripod so that the leg may come in 
contact with one of the iron supports. If the tripod has been 
painted, scrape the paint off. What happens when the contact is 
made? This is known as Galvani's experiment. 

287. Make another nerve-muscle preparation of the other hind 
limb, but cut the sciatic nerve as near to the myel as possible and 
separate the leg from the body at the femoro-pelvic joint. Remove 
the skin a far as the foot, With the forceps crush the gastrocnemius 
muscle near the tendon of Achilles. See that the end of the nerve 
is cut off squarely. With a small brush or thin glass rod lift the 
nerve very carefully in such a way that its cross-section may fall 
upon the injured portion of the muscle. This stimulates the nerve 
and causes a contraction of the muscle due to the so-called demar- 
cation currents. 



83 



XX 

THE INDUCTION COIL 



288. Induction Machine or Inductorium. Principle of 
action : If portions of the wires forming two separate circuits be 
placed parallel to each other, as in the case of the planes of two 
spirals or coils of the inductorium, the one wire primary (P), being 
connected with a source of electricity (battery), the other, the sec- 
ondary (S), being simply a closed circuit; whenever the P circuit 
is closed (made), or is opened (broken), currents will at those 
moments be induced in the S circuit. 




Fig. 13. Induction coil, ab, binding posts for single induced currents; cd, 
binding posts for interrupted currents ; e, post connecting Neef 's hammer with 
the primary coil; /, electro-magnet; p, primary coil; s, secondary coil. 

The make induction current flows in the S circuit in a direction 
opposite to that of the P circuit : whilst the break induction current 
flows in the same direction as the original battery current. These 
induction currents are of very short duration. 

Place the induction machine lengthwise in front of you on the 
table with the interrupter turned to the right. In the DuBois 
Reymond type the wires are wound into two separate coils : The P 

84 



coil which is supported by a woodlen upright attached to the base of 
the instrument is composed of relatively thick wire, while the S coil 
mounted upon a sliding foot is composed of very thin wire, in this 
case invisible, as it has a protective covering of vulcanite. 

The parallelism of the wire in the two coils is maintained as long 
as the axes of the coils coincide. The successive turns of the wire 
in each coil are also practically parallel to each other. The P coil 
is provided with a core of soft iron wire which magnetizes when a 
current passes in the surrounding wire, an electro-magnet being thus 
produced. 

The electrical field produced by the coil is greatly intensified by 
this core, and the effect on the S coil is correspondingly increased. 
The nearer the S coil is to the P coil, the more powerful will be the 
induction currents. 




/ 

Fig. 14, showing the direction of the make and break currents. In the sec- 
ondary circuit the direction of the break current is shown by the doted line and 
arrow, and the make by the continuous line. 

The electromotive force of the currents in the secondary bears 
a direct relationship to the primary. Thus, if there are 200 turns 
in the P and 6000 in the S, the eletromotive force of the induction 
currents would be about thirty times as great as the primary, inde- 
pendently of the influence exerted by the iron core. 

289. Connect the Secondary Circuit of the Inductorium. 
It is well to do this first in all cases. Fasten a key to the table close 
to the left end of the machine as it now rests upon the table, and 
connect the binding screws of the S coil with those of the key by 
means of two wires, so that when the key is closed the 'S circuit is 
thereby also closed. This is the short circuiting key in the secondary 
circuit. 

Now attach the long circuit wires by means of which the con- 
nection is to be established, with the seat of stimulation, i. e., attach 
the electrodes by their metal tags to the other pair of binding screws 
of the key. 

85 



290. Connect the P Coil for Single Induction Currents. 
Place the battery upon the table near the right hand end of the coil 
and attach a key to the table close to it; keep the key open. In 
making the connections always begin at the battery, and follow the 
direction the current will take. 





Fig. 15. — Key closed. 



Fig. 16. — Key open. 



Connect the C (carbon) pole of the cell to the key by a wire, 

then wire the other side of the key to the top binding screw, a fig. 13, 

of the P coil, wire b to the zinc pole of the cell. 

Withdraw the S coil to the end of the scale and let one co-worker 

hold the electrodes to the tip of the tongue, while the other makes 

the trials. 

Make and Break the P Circuit with the Key 1. 
Fig. 16. Do this smartly once or twice only and 
after each trial push the -S coil 1 centimeter towards 
the P coil. Let the co-worker indicate when he feels 
the "shock" and whether he does so at closure or 
opening. Note the position of the coil as soon as 
the minimal break shock is felt ; it is perceived first. 
Proceed with further trials until the make shock is 
also felt. Read off the position of the S coil. It is 
considerably nearer to the P coil. The break shock 
is the stronger of the two. Continue the approxi- 
mation of the iS coil by short distances to the P coil. 
The shocks will be stronger each time until finally 
unbearable. (Fig. 18). 

The strength of a stimulus can therefore be 

varied by changing the relative position of the S coil. It may 

approximately be assumed to change inversely with the square of 

the distance between the two coils. 




Fig. 17.— a, l, 
binding posts of 
primary coil. 



86 



Remove the core of soft iron from the P coil. Find the minimal 
shocks for break and make shock and compare with the readings in 
the previous experiments. 

Next take the S coil out of the slide and place it end on end 
close up to the P coil. Whilst making and 'breaking the P circuit 
turn the S coil so that its axis shall be ultimately set at right angles 
to that of the P coil. The shocks will rapidly diminish and finally 
disappear as the position of the S coil is changed. 



Battery, 




Fig. 18. — Apparatus as set up for make and break shocks. 

Explanation: When the battery current at the closure of the 
circuit is rising in strength in the primary, an opposing induction 
current is thereby generated in the P coil itself, which retards the 
battery current from attaining its full strength as soon as it other- 
wise would, and of course the effect upon the S coil is not so sudden 
a one. 

On breaking the P circuit an induction current 
is likewise generated which has the same direction 
as the disappearing battery current, and conse- 
quently it retards change of the electrical condition 
but does not interfere much with the suddenness 
of the subsequent drop in potential and therefore 
the effect upon the ! S coil is greater than at closure. 
291. Interrupted Shocks. Detach the wires 
from a and b and transfer them to the binding 
screws c and d. Adjust the top contact screw e 
jo that it touches the spring lightly. Fig. 13. 

On closing the P circuit this spring oscillates, 
automatically opening and closing the P circuit, 
and a succession of induction currents is generated 
in the S coil. The rate of their occurrence depends 
upon the length of the spring. 

87 




Fig. 19. — C, 
binding post ; E, 
primary coil ; D, 
electro - magnet 
and b i n di n g 
post. 



Explanation : The current from the battery flows through the 
binding- screw d, up the pillar through the spring, up through the 
top contact screw e'to the P coil, and thence round the electromagnet 
/ and back by the base of c to the battery. Fig. 13. 

When the current flows round the circuit, / is magnetized and 
draws down the spring thus breaking the top contact. Upon this 
the current stops flowing, the magnet ceases to act, the spring is 
released and again makes contact with e and so the circuit is re- 
established and the cycle begins anew. 

As the break shock is always the stronger of the two, it follows 
that if these shocks are passed through a tissue for some time that 
polarization effects will be set up. Ordinarily they are employed 
for a short time only, and this effect can be disregarded. 

292. Electrolysis of Po- 
tassium Iodide. An interest- 
ing example of electrolysis is 
is seen in the decomposition of 
potassium iodide. Dip a small 
Mrvt piece of filter paper in starch 






V»fttscZ« 



**L 



%C2 ^\^ paste to which about 5% of 

Fig. 20 



**$. potassium iodide has been 



added and lay the paper over 
the electrodes. Make and break the circuit using the single induced' 
current. Iodine is set free at the anode and turns the starch blue, 
forming the iodide of starch. This method may be used to deter- 
mine which is the anode. The direction of the current in the 
secondary coil of the inductorium may thus be recognized. (Porter) . 

A make contraction starts from the kathode, a break contraction 
from the anode. 

This experiment also shows that the current passes in opposite 
directions in make and break. 

293. The Break Extra Current. When a galvanic current 
traversing the primary coil of an induction machine is made or 
broken, each turn of the wire exerts an inductive influence on the 
others. When the current is made the direction of the extra current 
is against, or in an opposite direction to that from the battery, but 
at break it is in the same direction as the battery current. 

Apparatus: Battery, two keys, wires, primary coil of induc- 
tion machine. Arrange the apparatus according to the diagram. 

88 




FIG. 21.— The Break-Extra Current 



Fig. 21. Both keys and the coil are in the primary circuit, the keys 
being so arranged that either the primary coil, P, or the electrodes 

attached to key 2 can be short 
circuited. Test either by elec- 
trodes applied to the tongue or 
by means of a nerve muscle prep- 
aration. Close key 1, thus short- 
circuiting the coil. Open and 
close key 2. There is very little 
effect. Open key 1, the current 
passes continuously through the 
primary coil. Open key 2 ; a 
sensation is felt, due to the 
.break-extra current. 

294. Rheocord. The rheocord consists of a brass or G-erman 
silver wire (in this case 20 meters in length), placed along a square 
board, with its ends connected with binding posts. On the wire is 
a "slider" which can be pushed along the wire as desired. On 
account of the difference in potential of the two poles of the cell, 
the potential through the wire will fall uniformly from the anode 
to the kathode. The difference of potential between post and post 
1 will be practically one-tenth the electromotive force of the element. 
Connect the battery (or two) through a key with the rheocord, at 
posts and 1. Arrange the electrodes from the "slider" post to 
come in contact with the muscle. Close the key and note the effect. 
Move the ' ' slider ' ' along the wires and note the effect. 

An electric current can be graduated by changing the number, 
arrangement and size of the cells, or by using a rheocord to divide 
the current itself, the battery remaining constant. 

295. Unipolar Excitation. Set up the battery and induc- 
torium to give single shocks, and at first attach only one wire to 
the secondary coil. Prepare a nerve-muscle preparation and place 
it upon a dry glass plate, putting the single wire from the secondary 
coil under the nerve. Open and close the key in the primary circuit ; 
no contraction occurs. Insert a second wire in the other binding 
post of the secondary coil and attach its other end to a gas pipe thus 
connecting with the earth. A contraction will now occur on opening 
or closing the primary circuit. In the latter case, the amount of 

39 



current which passes through the earth and the glass plate is suf- 
ficient to stimulate the nerve. The short-circuiting key in the 
secondary circuit is therefore used in most experiments in order to 
avoid excitation of the nerve in this way. 

296. Polarization of Electrodes. If a pair of clean platinum 
wires be immersed in water, and a current sent through them for a 
time, it is found that both of the platinum terminals become covered 
with bubbles of gas. The one in connection with the negative pole 
of the battery is covered with hydrogen, and the other with oxygen. 
Upon the removal of the battery and connection of the electrodes 
with a galvanometer, a current will be demonstrated having a reverse 
direction to that first induced. This condition at the electrodes is 
known as polarization of electrodes. 




Polarization of electrodes. 



If a piece of fresh animal tissue connects the pair of wire 
electrodes, instead of the solution, the same polarization occurs. 
Chemical changes occur where the wires touch the tissue which can 
act in the reverse manner, and set up a small current if the battery 
be removed and the electrodes connected by a conductor. This acts 
as a source of fallacy in many experiments and is of much import- 
ance when a very excitable tissue, such as a nerve, is dealt with. The 
following experiment will illustrate polarization. 

Arrange the apparatus as shown in Fig. 22, open the key k2 and 
close the key, kl. The current is sent through the nerve and will 
polarize it. There is no contraction of the muscle while the current 
is passing. After one or two minutes, open kl, and then rapidly 
close and open k2, when contractions will occur, which are due to the 
closing and opening of the small current set up by the polarized 

90 



electrodes. The contractions diminish quickly in amount as the 
nerve becomes depolarized. 

In order to avoid polarization effects, special forms of electrodes 
may he used. These are known as unpolarizable electrodes and 
usually consist of a glass tube containing a saturated solution of 
zinc sulphate. The electrode end of the tube is filled up with a 
pad of china clay or earners hair brush, upon which the nerve is 
laid ; the other end of the tube is fitted with a binding post attached 
to a zinc wire which dips into the zinc solution. The electrodes in 
the moist chamber are an example of unpolarizable electrodes. 



XXI 
NERVE STIMULI 



297. Each student is to have a frog, which is to be pithed and 
have its brain and myel destroyed by passing a tracer or seeker 
through the spinal canal. The legs are to be used for nerve muscle 
preparations. Dissect one leg for the first series of experiments, and 
reserve the other leg for the second series. Begin the dissection 
upon the dorsal aspect of the leg, removing the skin and muscles 
very carefully until the sciatic nerve is exposed. Dissect out the 
nerve as far as possible and moisten frequently with the salt 
solution. Remove all of the muscles as far as the knee, leaving the 
femur and nerve entirely isolated. Avoid all injury to the nerve 
during dissection, and apply the normal salt solution every few 
minutes with a camel's hair brush. Arrange the nerve muscle 
preparation by placing the femur in a clamp and allowing the nerve 
to hang freely. A small lever may be pinned' to the foot to empha- 
size any movement that may occur. Apply the following stimuli : 

298. Mechanical. Pinch the free end of the nerve sharply with 
a pair of forceps ; the muscles contract and the foot is raised sud- 
denly. Cut off the pinched portion. Contraction again occurs. 

299. Thermal. To the same preparation apply at the free end 
of the nerve a wire or needle heated to a dull heat or a lighted match. 
Contraction again occurs. Cut off the dead part of the nerve. 

300. Chemical. Place some saturated solution of sodium 
chloride in a watch glass and let the free end of the nerve dip in it, 

91 



It requires a few minutes for the salt to diffuse into the nerve on 
account of the difference in the specific gravity. Soon the joints of 
the toes twitch and -by-and-by the whole limb is thrown into irreg- 
ular, flickering* spasms, which terminate in a more or less continous 
contraction, constituting tetanus. Cut off the part of the nerve 
affected by the salt ; the spasms cease. 

(a) Finish the experiment by exposing the nerve to the vapor 
of strong ammonia in a test tube or bottle. The ammonia must not 
act directiy upon the muscle, the tube should be raised slightly 
above the level of the muscle and the end of the nerve elevated to 
the mouth of the tube. There should be no contraction if the vapor 
has not come in contact with the muscle. Apply ammonia to the 
muscle. It contracts. 

301. Electrical. For the following experiments, use the other 
leg of the frog, taking the same precautions in the dissection and 
application of the salt solution. 

(a) Arrange the nerve-muscle preparation with the femur in a 
clamp and connect with a recording lever. Arrange the battery and 
DuBois Reymond Key with the induction coil. Connect the battery 
wires with the primary coil and remove the secondary coil to the 
lower end of the scale. Arrange the electrodes with a short-circuiting 
key and a recording drum with smoked paper, conveniently to the 
preparation. When all is in readiness, close the connection by 
means of the key by raising or lowering its lever ; or in other words 
"making" or "breaking" the curent. Gradually move the second- 
ary coil along the scale while making and breaking the current. The 
current is made when the connection is complete, and broken when 
the connection is interrupted. Note at what point on the scale the 
first result appears and whether it be from "make" or "break." 
Make a table of your results as follows : In one column indicate the 
distance of the secondary coil from the primary. In another, 
Response at Make, and the last column, Response at Break. This 
is electrical stimulation in the form of single induction shocks. The 
highest tracings represent maximal and the lowest minimal stimuli. 
Submaximal stimuli represent any strength between these two 
extremes. 

(b) Remove the induction coil and use only the battery with its 
wires and the key. Make and break the current as in (a). Notice 

92 



that if the key be so arranged as to permit the current to flow 
continuously through the nerve, no contraction occurs, provided 
there be no variation in the intensity of the current. Rapidly make 
and break the current by opening and closing the key ; a more or 
less perfect tetanus is produced. This is the constant current form 
of stimulation. 

(c) Remove the wires from the primary coil and connect them 
with the sockets leading to the vibrating hammer. On applying 
the electrodes to the nerve or muscle the latter is at once thrown 
into a state of rigid spasm or continuous contraction called tetanus. 
Compare the tracing with that of (a). This form of stimulation is 
known as the interrupted current or repeated shocks, 

302. Electricity itself is not readily conveyed through the 
nerve, but the irritation caused by it, generates a stimulus which is 
transmitted. Llgate the nerve by tying tightly around it a piece 
of thread. Stimulate the nerve as before ; there should be no result, 
as the ligature has crushed the nerve and blocks the passage of the 
stimulus. Scratch your name on the above tracings to identify 
them. The tracings may be made permanent by drawing the paper 
through a pan of shellac and allowing them to dry. 



XXII 

SECONDARY AND PARADOXICAL CONTRACTION. 
ERGOGRAPH 




Secondary 
Con I rcLctt oris 

Fig. 23. — n, nerve; m, 
muscle. 



303. Arrange the induction appara- 
tus for single make and break shocks. 
Pith a frog and use the hind legs for 
nerve-muscle preparations, and arrange 
them upon a glass plate. Place the 
sciatic nerve of the left leg upon the 
gastrocnemius muscle of the right leg, 
fig. 23. Place the sciatic nerve of the 
right leg over the electrodes and stimu- 
late the nerve with single induction 
shocks and note that the muscles of both 
the right and left leg contract. The con- 
traction in the left leg is called a second- 
ary contraction. Repeat the experiment, 

93 



using the constant current. Note if there is any difference between 
the make and break shocks. 

304. Secondary Tetanus. Prepare the induction apparatus 
for interrupted shocks, and again stimulate the right sciatic nerve. 
The right gastrocnemius muscle is thrown into tetanus. The left 
gastrocnemius is simultaneously tetanized. This is known as a 
secondary tetanus, and is a proof of the "action current" in muscle. 
The left sciatic is stimulated by the variation of the muscle current 
during the contraction of the right gastrocnemius. Li-gate the left 
sciatic near its muscle ; stimulate the right sciatic ; there should be 
no contraction of the left gastrocnemius. 

Leaving the left sciatic in position, tie a ligature around the 
right sciatic near its muscle and stimulate. Is there contraction in 
either preparation ? 

This experiment also shows that electricity, as such, is not trans- 
mitted through the nerve, although the thread of the ligature is a 
conductor. The electricity serves merely as a stimulus causing an 
impulse which can traverse the normal nerve but cannot pass beyond 
a ligature or a crushed portion of the nerve. 

305. Secondary Con- 



electrode 




traction From Nerve. 
Make a nerve-muscle prep- 
aration of the right hind 
leg of the frog and lay it 
on a glass plate. Dissect 
out a long piece of the left 
sciatic nerve. Remove 
and arrange in such a way 
upon a block of parraffin 
that one centimeter of it 
overlaps a corresponding 
length of the right sciatic, 
fig. 24. 
Stimulate the left nerve with a single induction shock ; the muscle 
contracts. Stimulate with the interrupted current; the muscle is 
thrown into tetanus. Stimulate also with the constant current and 
compare effects. If properly conducted this experiment will also 
show that a nerve impulse can pass in both directions. 

94 



jVeri/e ■tm.vtxlse 



bo ih directxo n s. 



Fig. 24. — n, nerve; m, muscle. 



Stimulate the left muscle directly by applying the electrode to 
it and note any effect upon the right muscle. 

Ligate the left sciatic nerve between the electrodes and the right 
nerve; stimulate again. The muscle does not contract, In the 
former case, therefore, its contraction was not due to an escape of 
the stimulating current. 




Fig. 25.—- Secondary contraction from the heart. 

306. Secondary Contraction From the Heat. Excise the 
heart ; lay the nerve of a fresh nerve-muscle preparation upon it as 
per diagram fig. 25. The muscle contracts at each beat of the 
heart, being excited by the electrical current which accompanies 
each beat. 

Crush the apex of the ventricle with the forceps and arrange 
the nerve so that its cut end will come in contact with the injured 
portion of the heart. Note the result. 

307. Paradoxical Contraction. Arrange 
the battery and key for a constant current 

Pith a frog and expose the sciatic nerve 
down to the knee. Trace out the two branches 
into which it divides, fig. 26. Cut off one of 
these branches as near as possible to the knee 
and stimulate near its cut end. The muscles, 
supplied by the other branch of the nerve con- 
tract. Try mechanical or chemical stimulation 
of the same branch. What is the result ? 

The second branch is stimulated by the 
electrotonic alteration of the first. 

Electrotonus — modification of the vital 
-m, nerve; m, properties of irritable and contractile tissues 
when influenced by a constant battery current. 
(Stirling). 

308. Experiment with Ergograph. Adjust the apparatus so 
that the lever will write upon a drum revolving at its lowest rate 

95 




Pa r-oLcCox. teat 
Contraction, 



Fig. 26 
muscle. 



of speed. Tie the three fingers of the right hand leaving the index 
finger free. Adjust this finger to the vertical rod connected with 
the lever. Raise and lower the finger thus causing a simultaneous 
movement of the lever, which leaves its record upon the smoked 
paper. Raise and lower the finger at regular intervals (one second) . 
The abductor indicis is the principal muscle involved. Continue 
until distinct fatigue occurs. Fix and preserve the tracing. 



XXIII 

ELASTICITY OF MUSCLE. INDEPENDENT IRRITABILITY. 
SARTORIUS EXPERIMENT 

309. Dissect out the gastrocnemius muscle from the frog's leg. 
Attach the femur firmly in the muscle clamp and the tendon to the 
writing lever, to which a small scale pan is attached. See that the 
lever writes horizontally on a stationary drum. The weight of the 
scale pan may be neglected. 

Place in the scale pan, successively, different weights; 10, 20, 
30, 40, 50, 60, 70, 80, 90 and 100 or more. Put in the 10 gram 
weight and allow to remain 30 seconds. The lever will descend. 
Remove the weight and the muscle will return to its original position. 
Replace the weight, the lever will drop along the line it first made, 
revolve the drum a very short distance horizontally and add 10 
grams more ; the lever will descend. Revolve the drum again for a 
short distance equal to the previous horizontal distance, add 10 
grams more and repeat the above processes until the heavier weights 
have been attained. The "steps" of the "staircase" will become 
shorter and if the apices of all of the "steps" be joined, the line 
will form a hyperbola. At the end of the experiment remove the 
weights and note any contractile phenomena. Does the muscle 
return to its original position ? 

Repeat the experiment with a thin strip of rubber, substituted 
for the muscle. Join the apices of the "steps" with a line and 
compare with that of the preceding experiment. 

Test in the same way the elasticity of a short strip of aorta (cat) 
provided for the experiment. 

Test also a short piece of the frog's intestine. 

96 



310. Independent Irritability of Muscle. Apparatus: Bat- 
tery, induction coil, two keys, wires, electrodes, curare, etc. 

Arrange the battery and induction coil for an interrupted 
current with one key in the primary circuit and the other key to 
short-circuit the secondary. 

Destroy the (brain of a frog. Expose the sciatic nerve and the 
accompanying artery and vein on the left side, being very careful 
not to injure the blood vessels. Isolate the sciatic nerve and tie a 
stout ligature around all of the other structures of the leg. 

Inject 5 to 10 drops of the curare solution into the abdominal 
cavity. The poison will be carried to every other part of the body 
except the leg below the left ligature. The animal is paralyzed in" 
from twenty to thirty minutes. If the non-poisoned left leg is 
pinched it is drawn up, showing that it has not lost its reflex powers ; 
while the poisoned right leg has lost its reflex. 

When the frog is thoroughly under the influence of the poison, 
i. e., when all reflexes cease, expose both sciatic nerves as far up as 
the vertebral column and as far down as the knee. 

! Stimulate the right sciatic nerve. There is no contraction. 
Stimulate the right gastrocnemius muscle ; it contracts. The poison 
has therefore not affected the muscle. 

Stimulate the left sciatic nerve above the ligature, the left leg 
contracts. The poison has not affected the nerve trunk. The nerve 
impulse is blocked by the curare, in all probability by paralysis of 
the end plates of the motor nerves within the muscle. Apply several 
dlrops of a strong solution of curare to the left gastrocnemius, and 
after a time stimulate the left sciatic nerve ; there should be no con- 
traction, but on stimulating the muscle directly, contraction occurs. 

311. Bernard's Method Two nerve-muscle preparations are made. The 

nerve of one (A) is immersed 20 to 30 minutes in a solution of curare in a 
watch-glass. The muscle of the other preparation (B) is immersed in the curare 
in another watch-glass for an equal length of time. On stimulating the nerve 
of A, its muscle contracts; on stimulating the nerve of B, its muscle does not 
contract, but the muscle contracts when it is stimulated directly. In A, although 
the poison is applied directly to the nerve trunk, the nerve is not paralyzed. 

312. Relative Excitability of Muscle and of Nerve. Deter- 
mine the minimal break shock which will cause a muscle twitch 
through the sciatic nerve, and then apply the same stimulus to the 
gastrocnemius muscle directly. It will not cause contraction, 

97 



Slide the S coil nearer to the P coil until the stimulus is strong 
enough to cause the muscle to contract, and note the difference in 
strength required. 

This experiment does not permit of the conclusion that the muscle 
possesses independent irritability, as the nerve terminations in the 
muscle are not excluded. (See Curare Experiment). 

313. Changes in the Excitability of a Nerve When Dying. 
Dissect out the sciatic nerve of a frog, but do not cut it from its 
connection with the my el. Place under its whole length a strip of 
thin rubber or a piece of waxed paper and keep the nerve moist 
with the saline solution. 

Carefully raise the nerve with the glass rod or camel's hair 
brush and explore it from one end to the other with minimal single 
induction break shocks, the effect of which have been tested first at 
the middle of the nerve, and note if there is a difference in excit- 
ability at any point. There is usually one or two such points. 
Locate them. 

Determine this by the change produced in the muscular effect, 
such as an increase, decrease or absence of contraction. 

Now cut the nerve at its spinal origin, and compare the excita- 
bility at the cut end with that at a point near the muscle. Repeat 
this from time to time. The cut end will soon show a greater 
excitability, which will decrease later until it is completely lost 

A dying nerve first rises and then falls in excitability and finally 
loses it all together. A nerve undergoing the process of dying be- 
comes for a time, more irritable for this reason. 

314. Dead Muscle and Nerve. Immerse a nerve-muscle 
preparation for a few minutes in warm water (40°C). Apply all 
of the preceding stimuli and compare results with those obtained 
from a normal preparation. 

315. Kuehne's Sartorius Experiment. Carefully dissect out 
the sartorius muscle, and to the tendon which attaches it to the tibia 
tie a fine thread. The upper end of the muscle may be freed from 
its attachment to the symphysis. Suspend the muscle with its upper 
end hanging downward and bring up under it a little glycerin in a 
watch-glass until the end of the muscle just touches the glycerin. 
Observe for a minute or two. No contraction should result. Cut 
off the end which has touched the glycerin and note that the muscle 
contracts as a result of the mechanical excitation. Again touch the 

98 



cut surface with glycerin and observe. If only about one millimeter 
has been cut off there is again no contraction. Cut off a fresh milli- 
meter of muscle and repeat as before. It will be found that when 
about three or four millimeters of the cephalic end have been cut 
away, on contact of the freshly exposed end with the glycerin, the 
muscle shows irregular twitchings and is at last thrown into a state 
of incomplete tetanus. 

This experiment should be completed by showing that if a 
gastrocnemius nerve-muscle preparation be made and the cut end 
of the nerve dipped into glycerin, the gastrocnemius is thrown into 
a similar series of irregular twitching. Nerve fiber is therefore 
excitable to glycerin. The same experiment may be tried upon a 
curarized muscle. 

The experiment on the sartorius muscle confirms the fact, as 
shown by histological examination, that no nerve fibers are present 
in the ends of the muscle ; for the same experiment shows the same 
results for two or three millimeters of the distal end of the muscle. 
Muscle fiber is not excited by glycerin and not until enough of the 
muscle was cut away to expose the nerve fibers in the muscle did 
the irregular twitchings occur. 



XXIV 
THE MUSCLE CURVE. MUSCLE WORK 

316. The Moist Chamber. Muscle and nerve tissue dry shortly 
after their removal from the body and the usefulness of an experi- 
ment is, sometimes, much hampered by this fact. In order to 
prevent drying, a moist chamber is employed. This apparatus 
consists of a glass cover fitting tightly over the myograph (muscle 
electrodes). A thread passing through an aperture connects the 
muscle with the recording lever. A few pieces of blotting paper 
wet with the saline solution placed in the chamber keeps the air 
moist. 

317. The Muscle Curve. If a stimulus of very short duration 
be applied to a muscle or its nerve, the muscle responds by giving 
a contraction of very short duration. This is termed a simple twitch, 

99 



The curve obtained from a muscle falls naturally into three 
parts : 

(1). From the point of stimulation (a) to the point of com- 
mencing contraction (b). This is known as the latent period. 
During this time there is no change in the length of the muscle. A 
muscle does not contract simultaneously all over, but the contraction 
starts at some one spot and then spreads in a wave-like manner over 
the rest of the muscle. Following an excitation at one spot, the 
fibers in that position contract, but do not at first lead to a move- 
ment of the recording lever but rather to a stretching of the 
remainder of the fiber, both above and below the point of contract- 
ing. The parts which have to be removed possess some inertia. 

c, ordinate 




GLbjClSfCL 



Fig. 27. — db, latent period; ~bc, period of contraction ; cd, period of relax- 
ation. 

(2) . From the point of commencing contraction (b) to the highest 
point of the curve (c). This is termed the period of contraction. 
The curve is at first convex to the abscissa, or base line, which means 
that the rate of contraction is at first very slow as seen by the acute 
angle which the first part of the curve makes with the abscissa ; it 
then rapidly increases as shown : by the increasing inclination to the 
abscissa, and very soon reaches a maximum rapidity. From this, 
again, there is another change in rate, this time in the reverse 
direction, for the curve becomes concave to the abscissa line and 
gradually shortening becomes slower until at last it ceases, when the 
tangent to the curve becomes parallel to the abscissa. 

(3). The third portion of the tracing is from the highest point 

(c) to the point (d) at which the lever again reaches the abscissa. 
This part is called the period of relaxation. The terminal point 

(d) is often a difficult one to determine with any accuracy because 
the lever does not come instantly to rest ; but as it always possesses 
some inertia, it oscillates for a time about a mean position which it 
ultimately reaches. 

100 



318. Arrange a nerve-muscle preparation in the moist-chamber 
so as to record its contraction upon the drum. Connect the battery 
with the induction coil introducing into the primary circuit a make 
and break key and an electro-magnet. Use also a short-circuiting 
key in the secondary circuit. Arrange the writing tip of the lever 
from the electro-magnet, so that it will write just below and on the 
same vertical plane as the muscle lever. Arrange also for a time 
tracing. Spin the drum at a fairly rapid rate 'by hand and use 
single induction shocks by breaking the primary circuit. The lever 
of the electro-magnet will indicate the instant the current is sent 
into the nerve-muscle preparation. (The lever rises and falls 
alternately as the current is made or broken). The muscle lever 
will rise just after that of the magnet. The tips of the two levers 
having started side by side from the same vertical plane, the differ- 
ence on the two abscissas between the rising point of the lever from 
the magnet and the rising point of the muscle lever, will be the 
approximate latent period. Verticals drawn through the two 
abscissas at the rising points of the two levers will be of use in 
determining more accurately the extent of the latent period. 

Verify as far as possible on the tracing, the preceding statements. 
Also obtain a curve from the make shock alone (short circuit the 
secondary coil when the break shock should occur). Vary the 
position of the secondary coil and compare the curves. Get a 
tracing of the contraction of plain muscle, by using a piece of the 
intestine or stomach of the frog. Compare. 

319. Amplification or Magnification. The amplitude of the 
tracing is measured by the ordinates ; it is the distance which sep- 
arates each point of the tracing from the line of the abscissa. When 
the primitive length of the muscle does not change as in the period 
of latent excitation, this distance equals 0, and the tracing is 
included with the line of the abscissa. When the muscle shortens, 
the tracing is raised above this line to a height relative to the degree 
of shortening. When the muscle elongates the tracing falls below 
this line to a certain extent. But as the muscle acts on a long lever, 
the changes in the length of the muscle are amplified on the tracing 
in a noticeable way. If, for example, the lever has a total length of 
150 millimeters and the tendon of the muscle is attached to a point 
15 millimeters on the axis of rotation of the lever ; each millimeter 

101 



ot muscle shortening will be produced on the tracing by a height 
(amplitude) of ten millimeters (1 centimeter). 

It is not difficult when one knows the length of the lever and the 
distance from, the point of attachment to the axis, to calculate the 
actual degree of muscular shortening. 

Amplitude depends upon the length of muscular fiber ; the longer 
the fibers of the muscle the longer the curve of amplitude. In 
general the amplitude increases with the intensity of the excitation, 
but there is a limit. 

Determine the amplitude in your tracing by measuring the 
length of the lever in millimeters ; then measure from the point of 
attachment of the muscle to the fulcrum and divide the total length 
by this and the result will give the degree of magnification of the 
shortening of the muscle. 

320. Work Done During a Single Contraction. Arrange a 
gastrocnemius to record on a cylinder, but record only the "lift," 
the eylinder being stationary; move the cylinder by the hand as 
required. On the lever under the muscle attachment place weights 
of known value. With each twitch the muscle lifts the weight, and 
thus does a certain amount of work which is easily calculated. 

(a). Measure the height of the tracing from the base line or 
abscissa. This is conveniently done by a millimeter scale. The 
work which is done (w) is equal to the load (1) multiplied by the 
height (h) to which it is lifted; w=lh. But, of course, a long lever 
being used the tracing is much higher than the actual shortening 
of the muscle. 

(b). To determine the exact amount of the lift, one must know 
the length of the lever and the ratio between its arms. Suppose 
the one to be ten times as much as the other, then the total work in 
gram-millimeters must be divided by ten. 

Try different weights always using the same stimulus. It will 
be found that at a certain point there will be a maximum contraction 
after which the contractions will become weaker, because of the 
greater load and fatigue. Calculate the amount of work done at 
the maximum contraction. 

321. Record of the Thickening of a Muscle. Prepare a 
nerve-muscle preparation and lay it on a glass plate, keeping it 
well moistened with the saline solution. Arrange the battery and 

102 



induction coil as before. Adjust the heart lever (such as used in 
recording the beat of the frog's heart) so that the vertical portion 
of the lever rests upon the belly of the muscle. Use the break and 
then the make shocks as before. Compare these curves with the 
others. The drum should revolve at its fastest rate. 



XXV 
VERATRINIZED MUSCLE. FATIGUE. TETANUS 

322. Influence of Veratrine on the Contraction of Muscle. 
Destroy the brain of a frog, and inject hypodermically four or five 
drops of a 1% solution of Sulphate of Veratrine or a idlrop or two 
directly in the muscle. When the frog is under the influence of the 
poison, cause a reflex act by mechanically stimulating the skin of 
the leg. The limbs are extended, and remain so for several seconds, 
due to the prolonged contraction of the extensors overcoming the 
flexors and thus causing extension of the legs. 

Arrange the induction machine for single shocks and make and 
break the primary circuit by means of the key. Short-circuit the 
secondary. Do not stimulate the muscle often as the veratrine effect 
diminishes with the activity of the muscles. 

Make a nerve-muscle preparation; on cutting the nerve notice 
the prolonged extension of the legs. 

Arrange the muscle lever to record its movements on a slow 
moving drum. Take a tracing. Note that the muscle contracts 
quickly enough, but the contraction is very high compared with that 
of a non-poisoned muscle, while the muscle relaxes very slowly 
indeed. The relaxation phase may last several seconds. The tracing 
may show an uneven curve due to irregular spasms of the muscular 
fibers. 

Take another tracing with a quick revolving drum, and a curve 
reaching the whole circumference of the drum may be obtained, 
or the drum may go around several times before the relaxation is 
complete. 

Note that if the "veratrinized" muscle be made to contract 
several times the effect passes off — only a simple twitch being 
obtained — but is re-established after rest. A high temperature 
also causes it to disappear. 

103 



323. Fatigue of Muscle. Arrange an induction coil for break 
shocks, i. e., adjust the strength of the stimulus so that only one, 
the break and not the make, will appear. Prepare a nerve-muscle 
preparation; use a long lever and a weight of 50 grams. Use a 
slow revolving drum on which to record the muscle tracings, so slow 
that the ascent and descent of the lever form merely one line. Break 
the primary current at regular intervals. 

Note the "staircase" character of the record, i. e. y the second 
contraction is higher than the first, the third than the second and 
so on for a certain number of contractions. After that the height 
of the contraction falls steadily so that a line uniting the apices of 
all of the contractions forms a straight line approximately. Note 
later that in the phase of relaxation the lever does not reach the 
abscissa (contracture). If the march of events be arrested, and 
time given for repose, then, on stimulating, the lift increases, but 
the effect lasts only for a short time. 

After the gastrocnemius muscle is thoroughly fatigued, cut 
across the middle of the muscle with a scalpel and test with litmus 
paper the area of the cross section thus exposed. Test in the same 
way a cross section of the sartorius or some other muscle which has 
not been fatigued. 

d e 

c 

..... 




Fig. 28. — Curve of tetanus. At the beginning ad, the individual contrac- 
tions are somewhat discernible; these disappear and the general level of the 
curve rises to e. At this point the stimulus was removed and the curve dropped 
quickly toward the base line. 

324. Tetanus. Prepare a nerve-muscle preparation. Arrange 
to record on drum with the smallest fan. 

Place a key in the primary circuit, also one in the secondary 
and wire for interrupted current. Adjust the special wire and 
weight in the vibrating hammer, so that it swings at the lowest and 
gentlest rate. Open the short-circuiting key in the secondary 
circuit. Make the current for very short intervals in the primary. 
Adjust the weight so as to get faster vibrations and compare. 

104 



Study the tracings. The first are indented hut gradually there 
is more and more fusion of the teeth until a curve unbroken by 
depressions is obtained. In the curve of complete tetanus the ascent 
is at first steep then slightly more gradual, speedily reaching a 
maximum, when the lever practically records a horizontal line par- 
allel to the abscissa. "When the current is shut off the descent is 
very steep at first, and towards the end very slow. 

The number of shocks required to produce tetanus depends on 
the animal, the muscle, and the condition of the latter; the more 
fatigued a muscle is the more slowly it contracts, and therefore, the 
more readily does fusion of contractions take place. A fresh frog's 
gastrocnemius requires about 27 to 39 shocks per second to produce 
tetanus. 

Eeplace the key in the primary circuit by a metronome ; connect 
the wires with the primary coil. Vary the rate of vibration of the 
metronome and oberve the effect on the muscle curve. Compare 
with the previous tracings. 



XXVI 

EFFECT OF TEMPERATURE AND LOAD UPON MUSCLE. 
AFTER LOAD. INDUCTION IN NERVE. 
TELEPHONE EXPERIMENT 

325. Influence of Temperature upon the Contraction of 
Muscle. Prepare a gastrocnemius muscle, leaving it attached to 
the femur. The sciatic nerve may be disregarded. Fasten the 
femur in the clamp on the under side of the cover of the "muscle 
warmer. ' ' Tie the end of a fine copper wire around the tendon of 
Achilles. Bring the other end of the wire through the opening in 
the bottom of the muscle warmer and bend the wire around the 
muscle lever, making sure that the wire connecting the tendon with 
the muscle lever is vertical. Connect the end of the fine copper wire 
with one of the binding posts of the secondary coil. Connect the 
other post of this coil with the binding post on top of the cover of 
the muscle warmer. Connect the battery with a make and break 
key to the binding posts of the primary coil of the inductorium for 
single induced shocks. Fill the outer chamber of the muscle warmer 
with crushed ice. Bring the writing point of the muscle lever 

105 



against a smoked drum and let the drum revolve at a fairly rapid 
speed. Insert a thermometer in the top of the muscle warmer and 
stimulate the cooling muscle at intervals of 5 degrees with a maximal 
break current. As the temperature falls, the contraction curve 
becomes longer and the muscle shows a tendency to contracture. 
Indicate the temperature upon each curve made. 

Place a fresh paper on the drum and let it revolve slowly. Adjust 
a flame under the arm of the muscle warmer and stimulate the 
muscle with a maximal break current at intervals of 5°. Indicate 
the temperature upon the curves as before. 

"The height of contraction is least at the freezing point of the 
muscle ( — 5 degrees). It rises from the freezing point to zero; 
falls from zero to 19 degrees ; increases to 30 degrees, which is the 
maximum ; from 30 to 45 degrees diminishes again, and at 45 degrees 
the frog's muscle usually enters into a state called rigor caloris; the 
muscle becomes opaque, inelastic, resistant to the touch, shortens 
very considerably and undergoes chemical changes of great im- 
portance. The duration of contraction lessens with the rising tem- 
perature, being least at 30 degrees. Above 30 degrees the duration 
remains practically unchanged. The latent period is increased at 
low temperatures, diminished at high. Above 30 degrees the excit- 
ability to electrical stimuli diminishes steadily ; it disappears almost 
entirely before rigor is reached. ' ' — Porter. 

326. The Influfnce of Load Upon the Contraction of 
Muscle. A muscle to which a load is suspended is said to be 
"loaded." If a muscle contracts against a small and constant 
resistance, so as to be extended by a constant force during its con- 
traction, the curve described by a light lever attached to it is termed 
"isotonic." If a muscle contracts against a, large resistance, e. g., 
a strong spring, so that it can shorten very little, the curve described 
by a lever attached to it is termed "isometric." The latter is flat- 
topped, i. e., it shows a period of maintenance at maximum con- 
traction. The muscle reaches its maximum tension sooner than its 
maximum shortening and maintains the maximum tension longer 
than the maximum shortening. As ordinarily employed, the 
myograph gives an isotonic curve. 

Arrange the apparatus as for a simple twitch. Arrange a nerve- 
muscle preparation and attach to a lever weighted only with the 
scale pan. Obtain a tracing as thus arranged . Increase the loacl 

106 



by adding a 10 gram weight and get tracing. Increase the load 
still further by adding 30, 50 and 100 grams respectively and com- 
pare the tracings. 

It is noticed that the latent period increases as the load increases ; 
the period of contraction also tends to increase ; the period of relax- 
ation is at first decreased but with heavier loads is increased; the 
heights of the contraction diminish progressively as the load in- 
creases. 

327. After Load. In the ordinary study of muscle twitch, the 
contractions have occurred whilst the load on the muscle was- as 
nearly as possible constant. There is, however, a method which is 
exemplified in many bodily movements, in which the muscle is under 
a low tension until it commences to contract, and then, only, exper- 
iences a rise of tension. This is called the method of after loading. 
Arrange the apparatus for taking a simple twitch with a nerve- 
muscle preparation. Load the muscle with a weight of 20 grams 
attached to the pully of the muscle lever. Have the screw of the 
muscle lever adjusted so that the muscle itself bears no weight. Now 
lower the screw so that the whole load is carried by the muscle and 
bring the writing tip of the lever so that it will write horizontally 
upon the drum. Get a tracing of the muscle curve, using the break 
shock only. Now raise the screw to support the writing lever, so 
that the writing point is placed at the level of the apex of the curve 
just taken. Record from this position another curve. It will be 
found that the muscle still raises the lever. Raise the screw again 
until the level of the writing point is at the summit of this second 
curve, and again get tracing. Repeat this process until no further 
result is obtained. 

The important and characteristic feature of these curves is that 
though the weight is supported at a level which it just reached at 
the height of a previous contraction, it is still further raised when 
the muscle is again stimulated. Under such conditions, the muscle 
contracts to a greater degree than when freely loaded. The latent 
period is increased as the height of support of the weight is in- 
creased. This is accounted for by the fact that the muscle is taking 
in any "slack" there may be; also that it is gradually increasing 
its tension until it is able to lift the load. The first portion of such 
a twitch is isometric ; but beyond a certain point it suddenly becomes 
isotonic, and its shortening is then registered. 

107 



328. Induction in Nerves. Remove the secondary coil and 
with the aid of a glass plate, lay the nerve of a well moistened 
nerve-muscle preparation upon the primary coil in such a way that 
the free end of the nerve touches the nerve near the muscle or 
touches the muscle itself, so as to form a dosed circuit. Make and 
break the primary circuit. Make and break currents will be induced 
in the nerve and the muscle will contract. 

329. Telephone Experiment. Arrange a nerve-muscle prep- 
aration with its nerves over a pair of electrodes. Connect the 
latter with a short-circuiting key. Attach the key to the telephone 
by means of two wires. Open the short-circuiting key ; shout or blow 
into the telephone, and note that the muscle contracts vigorously. 
Eemove the electrodes from the key and connect directly with the 
telephone. What is the result ? 

330. Form and Volume of a Contracting Muscle. Dissect 
out the gastrocnemius muscle of a frog. Connect the hooked elec- 
trodes at each end of the volume tube with the muscle. The tube 
is filled with saline solution which has been boiled and allowed to 
cool down to the temperature of the room. Replace the stopper 
in the tube in such a way that all air bubbles shall bp excluded. 
The height of the water in the capillary tube may be adjusted to 
the proper level by moving the glass rod in the stopper in or out. 
Connect the electrodes of the volume tube with the secondary coil 
and, using a single induction current, send a maximal break shock 
into the muscle. Note very carefully the level of the water in the 
capillary tube before, during and after the contraction of the limb. 
Does the level* of the water in the capillary tube change? 



XXVII 
VAGUS CONTROL OF THE HEART. ACTION OF DRUGS 

331. Cardic Vagus of the Frog or Turtle. Refer to the 
previous dissection of the vagus. Pith the animal. Lay it on its 
back. Expose the heart, remove the sternum and pull the fore legs 
well apart. Distend the oesophagus by introducing a glass rod or 
tube ; the nerves leaving the cranium are better seen winding from 
behind when the oesophagus is distended. (Frog). Remove such 

108 



muscles as may be necessary. Three nerves are seen coursing round 
the pharynx. The lowest is the hypoglossal, easily recognized by 
tracing it forward to the tongue, next is the vagus in close relation- 
ship with a blood vessel, and finally the glosso-pharyngeal. Observe 
the laryngeal branch of the vagus. The vagus as here exposed, 
outside the cranium, is really the vago-sympathetic as it contains 
fibers from the sympathetic system. The glosso-pharyngeal and 
vagus leave the cranium through the same foramen, in the exoc- 
cipital bone, and through the same foramen the sympathetic enters 
the skull. (Frog). 

Stimulation of the vagus. Adjust a heart lever so as to record 
the contraction of the heart upon a drum moving at a medium rate 
of speed. Place well-insulated electrodes under the trunk of the 
vagus, stimulate it with an interrupted current, and observe that the 
whole heart is arrested in diastole. 

If the stimulation is kept up the 
heart will finally "escape" from the 
influence of the stimulus and will 
recommence beating. Note if the 
auricles appear to be more inhibited 
than the ventricle. 

There is often a difference in effect 
between the two vagi. Sometimes one 
vagus is found not to possess any 
inhibitory fibers, in which case the 
opposite vagus is usually found espec- 
ially active. It is generally found 
that the effect is not identical on the 
two sides, one usually being more 
powerful than the other. 
The arrest, or period of inhibition, is manifest in the curve by 
the lever recording merely a straight line. If the laryngeal muscles 
contract, and thereby affect the position of the heart, divide the 
laryngeal branches of the vagus. 

There is an appreciable time or latent period, before the heart 
reacts to the stimulus and likewise when the stimulus is removed 
the heart does not at once regain its normal movement. Note that 
when the heart begins to beat again the beats are at first small and 
gradually rise to normal. In some instances, however, they are 

109 




I7c)u<fM 







Fig. 29. — Cardiac Nerves of 
the Frog. (Foster). 



more vigorous and quicker. Cut both vagus nerves and compare 
the tracing with those just obtained. 

332. Action of Drugs on Heart. The experiments may be 
performed upon a heart which has been removed and placed in a 
watch-glass or preferably upon the heart in its natural position. 

Muscarine. Pith a frog, expose the heart and with a fine pipette 
apply a drop of serum or saline solution containing a trace of 
muscarine, which rapidly arrests the rhythmical action of the heart, 
the ventricle being relaxed, i. e., diastole, and distended with blood. 
Get a tracing from the heart while under the influence of muscarine. 

Atropine. Eemove the solution of muscarine as much as possible 
by absorbing it with filter paper and after a few minutes, with 
another pipette, apply a few drops of a 0.5% solution of atropine 
sulphate in saline solution, the heart gradually again begins to beat 
rhythmically. Get a tracing. 

Pilocarpine. In another frog arrest the action of the heart by 
applying a few drops of a 0.5% solution of pilocarpine, and then 
apply atropine to antagonize it, and observe that the heart beats 
again after the action of the atropine. 

Nicotine. Apply a drop or two of a 0.2% solution of nicotine. 
Stimulate the vagus and note that it no longer arrests the heart's 
action, but stimulation of the sinus venosus does; so that nicotine 
paralyzes the fibers of the vagus and leaves the -intracardiac inhibi- 
tory mechanism intact. 



XXVIII 

THE SPHYGMOGRAPH, SPHYGMOMANOMETER, 
PNEUMOGRAPH 

In this exercise four styles of sphygmographs are available. 
Each student should obtain a tracing of his pulse from these instru- 
ments ; fix and preserve the records for comparison. 

333. Lud wig's Sphygmograph. With a soft pencil make a 
mark upon the skin of the wrist at the point where the radial pulse 
is most distinctly felt. Apply the instrument so that the button 
rests upon the pulse. Use the arm rest so that the parts will remain 
steady. Adjust the instrument so that the writing lever will move 
freely and give as large a curve as possible. Take tracings on the 

110 



revolving drum. Suspend respiration for a few seconds and notice 
whether there is any effect upon the pulse. 

334. Von Frey's Sphymograph. Adjust in the same manner 
as for Ludwig's. See that the clockwork runs properly and take 
tracings on the small drum. 

335. Richardson 's Sphymograph. Adjust the pressure of the 
button upon the artery until a maximum excursion of the marker 
is obtained. Wind up the clockwork, insert a strip of smoked paper 
between the guide wheels, and let the paper travel past the recording 
point as soon as the latter moves regularly. 

336. Teske's Sphymograph. Apply this instrument to the 
radial artery so that the amplitude of the pulse is at its greatest; 
When all is in readiness, set the smoked paper in motion by pressing 
the lever. Care must be taken not to move the instrument nor the 
arm of the person while the pulse is being registered. 

The instrument magnifies the movements of the artery fifty 
times. The clockwork is regulated so that the smoked paper shall 
pass through in ten seconds. Six times the number of pulsations 
traced on the paper will give the number per minute. The clock- 
work will not pass more than two lengths of the paper at the same 
rate. It is best to wind it up anew after two lengths have passed. 

337. Sphygmomanometer. The sphygmomanometer is an ap- 
paratus designed for the purpose of obtaining the blood pressure. It 
is a matter of some importance to know even approximately the 
arterial pressure and its variations under normal and abnormal 
conditions. The first practical method for determining this point 
upon man was suggested by von Basch (1887) who devised an 
instrument known as the sphygmomanometer. Since then a number 
of different instruments have been devised for the purpose. The 
brachial and radial arteries are the vessels ordinarily used, depend- 
ing upon the kind of apparatus. The general principle in the use of 
these instruments is to determine the amount of pressure necessary 
to obliterate the pulse and this can be observed from a registering 
apparatus which is a constituent of the instrument. The diastolic 
as well as the systolic pressures may be determined. 

The Riva-Rocci, Janeway, Stein and other forms of modern 
apparatus are available and give as up to date results as possible. 
Directions usually accompany the different makes and should be 
followed rather strictly to obtain accurate results. 

Ill 



338. The following information has been taken from literature 
accompanying the sphygmomanometer : 

Physiology of Blood Pressure. Arterial blood pressure is the 
pressure exerted by the blood column against the arterial wall at 
any specified point. 

As a matter of convenience the blood pressure values are usually 
taken on the arm over the brachial artery on man. On the horse 
they may be taken from the caudal artery in the tail. 

The arterial pressure is produced by the ventricular contraction 
of the heart forcing the blood out into the elastic and distensible 
arteries and propelling it forward against the resistance produced 
by the friction of the arterial walls and the increasing resistance 
produced by the lessening lumen of the capillaries. 

The blood flows with an intermittent character producing the 
pulse wave. 

During diastole the blood flows but with lessened force, the flow 
being maintained by the elastic contraction of the distended walls 
of the larger blood vessels. While the pumping force is intermit- 
tent a constant flow of blood is maintained to the organs. 

339. Systolic Pressure. At the end of the contraction of the 
left ventricle of the heart, systole, the highest blood pressure is 
present in the aorta and is designated as the systolic or maximal 
pressure. This represents the contracting power, or energy of the 
heart. 

340. Diastolic Pressure. At the end of systole the aortic valves 
close and the pressure is then maintained solely by the elastic recoil 
of the distended vascular walls. The pressure then gradually falls 
as the blood passes on through the capillaries until the end of 
diastole, when it reaches its lowest point. The pressure taken at 
this time is called the diastolic or minimal pressure. It represents 
the back pressure caused by the resistance from the constricted 
lumen and the larger surface area of the capillaries. The diastolic 
pressure indicates the amount of peripheral resistance the heart 
must overcome before it can propel the blood over the body. 

341. Pulse Pressure. By subtracting the figures of the minimal 
or diastolic pressure from those of the maximal or systolic pressure 
you obtain the pulse pressure. This represents the head of flow of 
the b,lood, In other words the pulse pressure gives the excess energy 
the heart exerts over and above the diastolic pressure, or peripheral 

112 



resistance. The pulse pressure indicates the amount of energy 
which actually carries on the circulation. 

All three pressures, systolic, diastolic and pulse pressure are 
important. 

While a great deal can he derived from an estimation of the 
systolic reading alone, it is equally important to determine the 
diastolic and pulse pressures. In many cases high or low systolic 
pressures are compensatory and in others what is apparently a 
systolic pressure within normal limits' may prove to be pathologic, 
when viewed in its relation to diastolic and pulse pressures. 




Fig. 30. The Janeway Sphygmomanometer. Manufactured by Chas. E. 
Dressier, New York City. A. Mercury manometer. B. Leather cuff, contain- 
ing rubber sac. C. Rubber bulb with deflating attachment. D. Removable 
extension of manometer. 

There is a reciprocal balance maintained under normal con- 
ditions between the systolic pressure and the diastolic pressure, 
through the vasomotor system. During disease this relation is dis- 
turbed and affords one of the best means of determining the 
condition of the cardiovascular system. 

342. Normal Readings. The pulse pressure should equal 

X13 



approximately one-third of the systolic pressure and the diastolic 
two-thirds of the systolic pressure, when a normal balance is main- 
tained. 

The general range of the pulse pressure, within normal limits, 
is from 25 to 40 mm. Hg\ 

Diastolic pressure ranges between 60 to 105 mm.Hg. 

Systolic pressure lies between 110 to 145 mm. Hg. 
Any sustained systolic pressure of 150 mm. Hg. or over is path- 
ologic regardless of age. A sustained systolic blood pressure below 
110 mm. Hg. should also be considered pathologic. 

While pulse pressure with corresponding normal systolic read- 
ings vary between 25 to 40 mm. Hg. the best way to determine the 
significance of the pulse pressure is by its relation to the systolic 
reading. If the correct percentage relation of the pulse pressure 
to the systolic pressure (thirty- three and a third per cent.) is present 
then a compensatory balance has been established, and even though 
the systolic reading be abnormally high the blood pressure should 
not be interfered with save by finding a cause for the hypertension 
and removing it, if possible. 

It is important to remember that a high diastolic pressure indi- 
cating a high peripheral resistance would require a correspondingly 
high systolic pressure to compensate for it. 

343. Summary. Systolic pressure represents the energy of the 
heart. 

The normal range in adult males is from 110 to 145 mm. Hg. 

In females the readings are about 10 mm. Hg. lower. 

In children from 91 mm. Hg. at three years to 105 mm. at fifteen 
years. 

Diastolic pressure represents the peripheral resistance and lies 
between 70 to 110 mm. Hg. 

Pulse pressure represents the head of flow which carries on the 
circulation. It is about thirty-three and one-third per cent, of the 
systolic pressure and varies from 25 to 40 mm. Hg. 

344. Auscultation Method. This method of taking blood pres- 
sure was originated by Karotkoff, of Russia, in 1906. It has to some 
extent replaced the palpation and oscillatory methods for clinical 
work. The accuracy, ease and. simplicity of the auscultation method 
have caused it to be used quite generally. 

114 



By this method Diastolic pressure is as easily taken as the Sys- 
tolic. 

345. Method, The pressure in the constricting arm band is 
raised above the obliteration of the pulse. Then slowly release the 
air and listen with a stethoscope over the artery about one inch below 
the cuff. Five phases are noted : 

1. At first there is no sound heard but in a short time a sharp 
clear thumping sound becomes audible (first phase). The first 
thump is the time of the systolic pressure. 

2. A murmur follows the tapping sound (second phase). 

3. The murmur in turn is replaced by a second tapping sound 
(third phase). 

4. The tapping sound more or less abruptly becomes dull 
(fourth phase). This change in the character of the sound indi- 
cates the time of the diastolic pressure. 

5. Shortly after the fourth phase all sound disappears (fifth 
phase). 

The beginning of the first phase is recognized as representing 
the correct systolic period and the beginning of the fourth phase as 
the correct diastolic period. 

346. Precautions. Care should be taken to see that the pneu- 
matic cuff is snugly applied to the bared extremity, the patient 
should be in a relaxed, comfortable position, and the cuff on a level 
with the heart. Mental relaxation is important and the patient 
should also have been physically quiet for a short time before obser- 
vation. Subsequent observations should be taken with the patient 
in the same position and about the same time of day with relation 
to meals, etc. Mental excitement, exercise, meals, edema, and 
dyspnea all tend to cause hypertension and should be eliminated in 
taking observations. Eeadings should also be taken as rapidly as 
possible, as prolonged pressure affects their accuracy and is un- 
pleasant to the patient. 

347. The Pneumograph. Apply this apparatus to the thorax. 
Connect with a tambour and take a tracing on a revolving drum. 
Study the curves. Does expiration occupy a longer time than inspi- 
ration? Compare with other members of the group. 

Do not look at a tracing while it is being made, a person un- 
consciously attempts to regulate the breathing so that a perfect 

115 



curve is made. Note the effect upon the curve of the following 
phenomena : squeeze suddenly a rubber bulb held in the right hand ; 
swallow a few mouthfuls of water ; hold the breath while you count 
ten; speak a few words; laugh. Have your co-worker indicate on 
the drum when each of these acts are performed. Use a time 
marker marking half seconds. 



XXIX 
THE CIRCULATION 



348. The Scheme op the Circulation. The circulatory system 
may be considered as consisting of a contractile organ — the heart, 
connected on one side with a system of elastic vessels: — the arteries, 
which expand into a very large area of thin-walled capillaries. The 
capillaries converge to form the venous system which connects with 
the opposite side of the heart. The arteries possess thicker walls 
than the other vessels and if a cut be made into an artery, especially 
a large one, the blood issues in spurts and with considerable force. 
If the bleeding is to be checked, pressure must be made between the 
cut and the heart. The veins are thinner walled and are usually 
more or less collapsed. If a cut be made into a vein the blood may 
issue in considerable amount in a constant stream but without much 
force. If the flow is to be checked pressure should be applied be- 
tween the cut and the capillaries. 

If the flow of a fluid through a tube be examined it will be 
found that the longer the tube the greater is the surface for friction 
and that the resistance is increasingly greater as the diameter of the 
tube diminishes, so that in the actual circulation the resistance in 
the very small arteries and capillaries is very great and this per- 
ipheral resistance in connection with the action of the heart must be 
considered a very important factor in regulating the flow of blood 
through the total circulation. If a fluid be passed through a tube 
with rigid walls and uniform diameter, it will be found that exactly 
as much fluid passes out as comes in and if the fluid be added 
intermittently the outflow will also be intermittent. This will also 
be true of a rubber tube if the diameter is uniform and its length 

116 



not too great. If, however, the end of the rubber tube is constricted 
or resistance be introduced, then the flow will lose its intermittent 
character and become more or less continuous. This is because a 
smaller volume can now escape in a given time and because the 
elastic nature of the walls permit them to expand to accommodate 
the extra fluid and as the recoil of the walls upon the fluid occurs 
immediately after the expansion the fluid is therefore forced steadily 
but continuously through the constricted orifice until the walls have 
returned to their original size. 

349. Many of the important phenomena of the circulation may 
for all practical purposes be worked out nearly as well upon an 
artificial scheme consisting of a bulb syringe and rubber tubing, as 




Fig. 31. — Scheme of the circulatory system. 



upon the circulation itself. In the accompanying diagram, H repre- 
sents the left ventricle of the heart with the mitral and semi-lunar 
valves. A, the arterial system with A M, a mercury manometer to 
show arterial pressure. C, the capillary system represented by a 
larger glass tube filled with small pieces of sponge or glass wool to 
imitate peripheral resistance by retarding the passage of the fluid. 

117 



C T is a connecting tube 'between the arterial and venous systems 
to simulate the arteries and capillaries in a dilated condition. CI, 
clamp. When this is closed the fluid must pass through the per- 
ipheral resistance in the capillaries C. V, venous system with V M 
its mercurial manometer for recording venous pressure, r is a con- 
nection which joins the venous system with that portion (auricle) 
which conveys the fluid to the ventricle. 

350. Disconnect the apparatus at r and fill the system with 
fluid by allowing the disconnected portions to drop in a basin of 
water and pumping the syringe. Continue until all of the air is 
•displaced. To insure this it may be necessary to raise the end of 
the venous tubing so that the air bubbles may escape. Support the 
end of the venous tube upon the edge of the basin so that the 
character of the outflow may be observed. See that the clamp on 
C T is open. Compress the bulb at slow intervals and note that the 
outflow is intermittent and occurs with each contraction of the bulb 
also that the mercury in the venous manometer oscillates with each 
contraction quite as freely as that in the arterial except for the 
slight friction and expansion of the walls, the conditions are very 
similar to those of rigid tubes. 

351. Screw up the clamp on C T thus forcing the fluid through 
the peripheral resistance of the capillaries. When the bulb con- 
tracts note that there is a sudden and marked rise of the mercury 
in the arterial manometer while that of the venous manometer does 
not rise but may fall a little, indicating a high arterial but low 
venous pressure. Note the pulsation in the arterial manometer and 
tubing and the complete or almost complete absence of it on the 
venous side. Note also that the outflow has lost its intermittent 
character and streams continuously between the heart beats. If 
no more contractions of the bulb occur the mercury of the arterial 
manometer sinks gradually to its former level and the pressure be- 
comes equalized. 

352. In the body the circulation is a closed system of tubes. 
Connect the apparatus at r, seeing that the system is full of fluid 
and carefully exclude air bubbles. There should be a slight positive 
pressure and the mercury of the two manometers should be at about 
the same height. Compress the syringe as before with the clamp 
closed so that the fluid must pass through the capillary system. 

118 



There will 'be a rise in the arterial and a fall in the venons mano- 
meter. If there is hut one contraction of the syringe, the pressure 
will gradually become uniformly distributed and the mercury of the 
two manometers arrive at the same level. 

353. If the contraction of the bulb be carried on at a definite 
rate the mercury will continue to rise in the arterial manometer 
until a point is reached when it will rise no higher but merely 
oscillates with each contraction and expansion of the ventricle. This 
is known as the mean pressure. The maintenance of pressure at a 
mean height means that this pressure including the action of the 
heart is just sufficient to force out through the peripheral resistance 
during the time of one complete cycle, exactly the same amount of 
fluid as comes into the arteries with each contraction of the bulb. 
If the venous manometer be observed it will be found that the 
mercury in the free arm falls with the expansion of the bulb as it 
returns to its original form. Note that the height of the arterial 
pressure can be changed: (1) by changing the rate or strength of 
the heart beat, (2) by changing the peripheral resistance, i. e., by 
opening or closing the clamp el. 

354. Effect of gravity. Let the heart H with its tubes hang 
at a lower level than the rest of the apparatus. There will be a fall 
in arterial and venous pressures, the vein v becoming more distended 
under the influence of gravity, by the pressure exerted by the 
column of fluid between it and the highest part of the apparatus. If 
the heart is not working the pressures in the two manometers may 
become negative. Thus, if the heart ceases to beat with an animal 
in the upright position, the blood of the body will tend to drain into 
the dependent parts of the venous system. 

355. Pulse. With the heart beating rhythmically, so as to 
maintain an average pressure on the arterial side, press the finger 
upon the rubber tubing representing the arteries and note the pulse ; 
tracings may be taken by placing a lever upon the tubing and 
recording the effect upon a kymograph or by the use of a sphygmo- 
graph. The beat of the heart should be maintained as regularly as 
possible. Tracings may be taken with the clamp cl, closed; with 
the clamp slightly opened and with the clamp fully opened. Trac- 
ings may thus be obtained corresponding to the high, medium and 
low tension pulse. 

119 




Fig. 32. — Scheme of the lymph and circulatory systems. 



356. Scheme of the combined blood and lymph systems: As 
arranged the circulatory portion is essentially the same as in the 
preceding scheme. The capillaries are represented as dilated. A 
clamp on V may be adjusted to regulate the pressure in the capil- 
laries. 

The scheme endeavors to represent the tissue or lymph spaces of 
the body and their relation to the capillary and lymph vessels ; how 
the lymph may be produced and how the lymph again reaches the 
blood. 

The small jar represented by Is is filled moderately full of small 
pieces of sponge to represent the tissues, while the spaces between 
represent the tissue or lymph spaces Is. The vertical tubing 1 emerg- 
ing from the middle of the jar is a lymph vessel which has its origin 
in the tissue spaces of the body. Vessels convey the lymph from 
the tissues to the thoracic duct through which it passes to enter the 
venous system. 

357. To work the apparatus, disconnect the venous system at 
r and let that portion attached to the heart be submerged in a beaker 
of water. Compress the bulb, until the circulatory apparatus is 
filled and the air displaced. The small pieces of sponge in the jar 
should have been previously dampened and the jar filled with water. 
Depending upon the force and frequency of the heart beat and the 
amount of peripheral resistance at el the amount of pressure in Is 

120 



will vary. If the jars Is and re are thoroughly tight, then when 
increased pressure occurs in the capillaries some of the fluid will 
pass out through the capillary walls to the tissue spaces ; the pres- 
sure in the latter will increase with that of the former. So that if 
the jar is perfectly tight and the pressure sufficient some of the fluid 
in Is will be forced through the openings of the beginning lymph 
vessel 1 and finally reach re the receptaculum chyli of the thoracic 
duct. If perfectly tight the pressure in re also rises and the fluid 
is passed on through td, the thoracic duct to enter the venous system. 
Sometimes if the air has not all passed out or the pressure not quite 
strong enough the fluid hesitates about passing through bl. In this 
case if bl be disconnected near re and a little suction applied the 
fluid will usually pass over quite readily. Reconnect and if a sim- 
ilar trouble occur between re and the venous systems try the same 
plan. Increasing or changing the pressure by regulating the clamp 
cl will oftentimes be sufficient. 

358. After the apparatus has been practised upon, the venous 
system may be connected at r, being careful to exclude all air. This 
would Simulate very closely the actual conditions as they exist in 
the animal body. By the use of manometers as in the previous 
experiments the differences in pressure may be observed in the 
arterial, venous and lymph systems. The pulse may be shown to be 
absent from the lymph as well as the venous system by the use of 
sphygmographs or writing levers. 



XXX 

BLOOD PRESSURE. STANNIUS' EXPERIMENT. MAXIMUM 

CONTRACTIONS. STAIRCASE CONTRACTIONS. MOTOR 

CENTRES. TEMPERATURE. ISOLATED APEX. 

INTRACARDIAC INHIBITORY CENTER 

359. Curarize a frog lightly, and expose the heart with the 
aortae leading off from it. Get ready a fine cannula with a short 
piece of rubber tubing attached. Fill the tubing and cannula with 
a 1 per cent solution of sodium carbonate ;and close the end of the 
tube with a clamp. Dissect out one of the aortae and tie a ligature 

121 



around it as far as possible from the heart. Pass a second ligature 
around the same aorta, without tying, nearer to the heart. Lift the 
aorta with the second ligature and with a pair of sharp pointed 
scissors make a slight incision in the vessel and introduce the cannula 
into the incision and tie it with the second ligature. Fill the prox- 
imal end of the manometer with a 1 per cent solution of sodium 
carbonate seeing that all air is excluded, so that when the tubing is 
attached to the manometer, there will be a continuous volume of the 
sodium carbonate solution from the cannula to the mercury of the 
manometer. Before attaching the tubing to the manometer, clamp 
the aorta or have your co-worker compress it carefully with a pair 
of forceps. Place the frog-board on a wooden stand, so as to bring 
the heart to a slightly higher level than the level of the mercury in 
the manometer. Bring the writing point of the lever of the mano- 
meter against a smoked drum and revolve the drum once so as to 
record a line of atmospheric pressure. 

After the cannula in the aorta, with its tube has been attached 
to the manometer, remove the clamp or forceps from the aorta and 
allow the blood from the heart to pump against the sodium carbonate 
and mercury in the manometer. The columns of mercury in the 
proximal and distal tubes will be no longer at approximately the 
same level. The mercury in the proximal tube will fall slightly and 
will rise correspondingly in the distal tube. Note that with each beat 
of the ventricle the column rises a short distance above the mean 
level and sinks again. Get a tracing of this blood pressure curve 
upon a very slowly revolving drum. The actual pressure, in milli- 
meters of mercury, is obtained by multiplying the mean height of 
the curve, above the atmospheric line, by two. Similar or more 
satisfactory results may be obtained from the carotid artery of the 
dog or cat, using a half saturated solution of magnesium sulphate 
instead of the sodium carbonate. 

360. Stannius' Experiments on the Frog's Heart. Some of 
the early and important experiments relating to the beat of the 
frog's heart were performed by Stannius, and bear his name. • 

If the sinus venosus is separated from the rest of the heart by a 
ligature of thread passed under the aorta and drawn tightly around 
the sinus at its junction with the auricle, the sinus venosus continues 
to pulsate, but the auricles and ventricles are quiescent. If the 

122 



auricles are now separated 1 from the ventricle by a thread ligature 
tied around the auriculo-ventricular groove, the auricles remain 
motionless, but the ventricle begins to beat, so that the sinus venosus 
and ventricle are pulsating, but with a different rhythm, while the 
auricles are at rest. The rate of the ventricular beat is usually 
much slower than that of the sinus. 




Fig. 33.— Aur, auricle; V, ventricle; S V, Sinus Venosus. The figure to 
the left shows the application of the ligature between the sinus and the auricle. 
In the figure to the right there is shown the second ligature between the auricle 
and ventricle. 



The quiescence of the auricles and ventricle, in the first case, 
has been supposed to show that the motor centers for the entire 
heart reside in the sinus, and that from them the motor impulses 
originate which keep up the rhythmical pulsations of the organ. 
But the fact that the ventricle begins to pulsate on its own account, 
as in the second case when separated by another ligature from the 
auricles, seems to show that it also contains motor centers. The 
hypothesis has been advanced that both sinus venosus and ventricle 
contain motor centers, while the auricles contain inhibitory centers. 

So long as the auricles are in connection, both with the sinus 
venosus and the ventricle, the motor centers in the latter two parts 
are supposed to be sufficiently powerful to overcome the resistance 
offered by the inhibitory centers, and thus the cardiac rhythm is 
maintained. When the motor centers of the sinus are removed, the 
inhibitory centers of the auricle are supposed to be so powerful as 
to keep both it and the ventricle in a state of rest. 

361. Cardiac Delay or Latent Period of Cardiac Muscle. 
In the case of skeletal muscle, the muscle is at rest and a stimulus 
excites it to contraction ; cardiac muscle has the power of contract- 

123 



ing rhythmically ; it will, therefore, be necessary to stop the heart- 
beat by the application of a "'Stannius" ligature. 

Arrange the apparatus for single induced shocks, and include in 
the primary circuit a signal magnet to mark the exact time the 
stimulus is applied. Use also a time marker recording in half 
seconds. 

After exposing the heart apply the ' ' Stannius ligature ' ' to stop 
the beat. Attach the apex of the ventricle to the heart lever. 
Arrange the three levers, heart, signal magnet and time marker so 
that their writing points will all be exactly in the same vertical line. 
Let the drum revolve. Stimulate the ventricle with a single induced 
shock. When the circuit is made or broken the lever of the signal 
magnet will immediately respond and shortly after the heart lever 
will also respond. The interval represents the "latent period" and 
may be about half a second, depending upon temperature and other 
conditions. 

Stimulate an auricle in the same way and note the longer 
"delay,-" the wave of contraction travelling slowly and delaying at 
the groove. 

Compare the cardiac latent period with the latent period of 
skeletal muscle. 

362. Maximum Contractions Only. Find the weakest stim- 
ulus that will cause contraction of the ventricle. Increase the 
strength of the stimulus but do not stimulate more than once in 
ten seconds, otherwise "staircase" contractions may result. The 
force of the ventricular contraction will remain the same in spite of 
the stronger stimulus. If the heart is capable of responding at all 
it will, in each case, give a maximum contraction. Stimulate either 
auricle in the same way and note the result. 

363. Staircase Contractions of the Heart. Apply the first 
Stannius ligature over the sino- auricular groove. Connect the 
apex of the heart with the heart lever. Record on a slowly moving 
drum. Stimulate the quiescent heart with single induction shocks 
at intervals of five seconds. Notice that the second beat is higher 
than the first, the third than the second and so on until a maximum 
beat is obtained. This is the "staircase" of Bowditch. 

364. Location of Motor Centers in the Frog's Heart. 
Dissect out the entire heart of a frog and note that it continues to 

124 



beat. Cut the heart vertically into three pieces, so that the middle 
portion will contain the auricular septum, in which lie the 
ganglionic cells. This portion continues to 'beat while the right and 
left lateral parts do not beat spontaneously, but will respond with 
a single contraction if stimulated. 

365. Effect of Temperature Upon the Heart Beat. . Leav- 
ing the heart in its usual position insert a glass tube into the 
oesophagus and allow it to project through the stomach. Pass water 
at different temperatures through the tufoe and note the number of 
beats in each case. 

366. Isolated Apex. Bernstein 's Experiment. Tie a ligature 
around the ventricle about half way between its apex and base, or 
compress it with a clamp or pair of forceps, the object being to 
destroy physiological continuity but preserving anatomical con- 
nection. The physiologically isolated apex does not contract. This 
would seem to indicate that the adult heart muscle is incapable of 
spontaneous rhythmical contraction. If the fbulbus arteriosus is 
compressed, the pressure of 'blood in the ventricle rises and is usually 
sufficient to stimulate the apex strongly enough to start it beating 
again. Remove the ligature and apply the presure to the bulbus 
and note the effects. 

367. Intracardiac Inhibitory Center in the Frog. Expose 
the heart of a frog, divide the frenum and tilt the heart upward to 
expose the whitish V-shaped crescent between the sinus venosus and 
right auricle. Stimulate the crescent, using fine electrodes, with an 
interrupted current ; if the current is sufficiently strong, the auricles 
and ventricle, after a brief delay, will cease to beat for a time, but 
they begin beating again even in spite of continued stimulation. 
Stimulate the auricles ; there is no inhibition. 

Connect the apex of the ventricle with the heart lever. Use a 
signal magnet marking seconds, in the primary circuit. Its lever 
will vibrate when the circuit is closed. Arrange so that its writing 
point will write immediately under and in the same vertical plane as 
the writing point of the heart lever. Get a tracing of the normal 
beat, then stimlate the crescent for one or two seconds as before. 
Inhibition results. After a pause the beat begins again, the con- 
traction passing as a wave from the sinus, through the auricles to 
the ventricle. 

125 



Stimulate the auricles. Note any effect upon the tracing. ( Dur- 
ing inhibition the sinus beats, but the auricles and ventricle do not, 
because the excitability is so lowered that they do not propagate the 
excitatory process) . 

Stimulate the ventricle mechanically, the heart beats in the 
reverse order from ventricle through auricles to sinus. 

Apply a few drops of atropine solution to the heart and again 
stimulate the crescent. There is no inhibitory effect as the atropine 
paralyzes the inhibitory fibres. 



126 



